1 


THE  DEVELOPMENT  OF  A METHOD  FOR  THE  DE 
TERMINATION  OF  THE  AMINO-ACIDS 
OF  FEEDS 


BY 


THOMAS  SHERMAN  HAMILTON 
B.  S.  University  of  Illinois 
1917 


THESIS 


Submitted  in  Partial  Fulfillment 
for  the  requirements  of  the 
Degree  of 


MASTER  OF  SCIENCE 
IN  CHEMISTRY 

IN 


THE  GRADUATE  SCHOOL 


of  the 


UNIVERSITY  OF  ILLINOIS 


1921 


UNIVERSITY  OF  ILLINOIS 


THE  GRADUATE  SCHOOL 


Decamber  eL2j ] 92 i 


I HEREBY  RECOMMEND  THAT  THE  THESIS  PREPARED  DNDER  MY 

SUPERVISION  BY Thomas  Sherman  Ha:iiilton 

ENTITLED  The  Deve lopment  of  a Metho 

of  the  Amino-Acids  of  Feeds 

BE  ACCEPTED  AS  FULFILLING  THIS  PART  OF  THE  REQUIREMENTS  FOR 
THE  DEGREE  OF Master  of  Science  in  Chemistry _ , 


Recommendation  concurred  in* 


Committee 

on 

Final  Examination* 


^Required  for  doctor’s  degree  but  not  for  master’s 


Digitized  by  the  Internet  Archive 
in  2015 


https://archive.org/details/developmentofmetOOhami 


-i- 


TABLE  OF  COITTEOTS 


THE  DEVELOR.IENT  OF  A J.TETHOD  FOR  THE  DETERI/.niATlOlI  OF  THE 
AlvIIHO-ACIDS  OF  FEEDS 

page 

I.  INTRODUCTION  1 

II.  HISTORICAL ^ 

III.  DEVELOPIviENT  OF  THE  METHOD 6 

A,  Extraction  of  the  Norqprotoin  Nitrogen  - --  --  --  --  - 7 

(1)  Extraction  vdth  anhydrous  ether  - --  --  --  --  7 

Experiment  I.  The  completeness  of  extraction 

with  ether  by  the  centrifuge  bottle  method  - ^ 

(h)  Extraction  with  cold  absolute  alcohol  ------  9 

Experiment  II.  Comparison  of  the  amounts  of  nit- 
rogen extracted  by  extractions  on  the  Buch- 
ner fumel  and  by  the  centrifuge  bottle 
method  -------------------  9 

(3)  Extraction  with  cold  1.0  per  cent,  trichlorace- 

tic acid  - --  --  --  --  --  --  --  --  - 10 

Experiment  III,  Comparison  of  the  amounts  of 
nonprotein  nitrogen  extracted  b;/  cold  1.0 
per  cent,  trichloracetic  acid  and  by  cold 
v;ater  and  0.02  per  cent,  hydrochloric  acid  - 12 

Experiment  IV.  Determir^ation  of  the  proper 
strerjgth  trichloracetic  acid  to  use  for 
the  extraction  of  nonprctein  nitrogen  - - - I3 

(4)  Separation  of  protein  and  nonprotein  nitrogen  - - 14 

Experiment  V.  Determination  of  the  amount  of 
colloidal  iron  necessary  to  precipitate 
the  proteins  from  the  nonprotein  extract  - - 17 

Experiment  VI.  Determination  of  suitable  con- 
ditions for  the  precipitation  of  proteins 
from,  the  nonptotein  extract  v.dth  colloid- 
al iron  ------------------  19 

(a)  Neutral  method  -----------  19 

(b)  Direct  method  - --  --  --  --  --  - 19 


-ii- 


page 


(c)  After  decomposition  of  trichlorace- 
tic acid  by  boiling  --------  19 

(dj  By  the  use  of  a buffer  substance  - - 20 
(e)  Second  precipitation  - --  --  --  - ^^0 

Experiment  VII.  Does  colloidal  iron  precipi- 
tate nonprotein  nitrogenous  compounds?  - - - 22 

Experiment  VIII.  Does  dilute  trichloracetic 

acid  hydrolyze  proteins?  ----------  23 

(5)  ProcedTui-e  finally  adopted  for  the  extraction  of 

. the  nonprotein  fraction  - --  --  --  --  - 23 

B.  Treatment  of  the  Residue  Insoluble  in  Ether,  Absolute 

Alcohol,  and  Cold  1.0  per  cent.  Trichloracetic  Acid  - 2li 


Experiment  IX.  Determination  of  solvents  and 
methods  of  extractions  necessary  to  com- 
pletely extrs.ct  the  nitrogenous  compounds 
from  the  residue  insoluble  in  ether,  alc- 
ohol, and  cold  1.0  per  cent,  trichlcrace- 


tic  acid  -----------------  2o 

C.  Extraction  of  Starch  with  Trichloracetic  Acid  ------  iiS 

Exp er indent  X.  Test  extraction  of  starch  with 

trichloracetic  acid  ------------  3^ 

Exper iment  XI.  Comparison  of  the  nitrogen  ex- 
tracted along  with  the  starch  by  hot  0.1 


per  cent,  hydrochloric  acid  and  by  hot  2.0 
per  cent,  trichloracetic  acid  ------ 

Exper inient  XII.  (a)  Doss  the  second  addition 
of  two  volumes  to  the  hot  2 per  cent,  tri- 
chloracetic acid  extracts  cause  any  addi- 
tiorjal  precipitation  of  starch?  (b)  Can 
the  proteins,  dissolved  by  the  hot  2 per 
cent,  trichloracetic  acid,  be  completely 
hydrolyzed  by  boiling  for  3 hours  with  5 


per  cent,  hydrochloric  acid?  -------  33 

(1)  Final  procedure  for  the  extraction  of  starch  - - - 37 

IV.  EXTRACTION  OF  STARCE  AND  NOiJPROTEIN  NITROGENOUS  CONSTITUENTS 

WITH  HOT  2 PER  CE1\IT.  TRICHLORACETIC  ACID  AND  THE  SEPARATION 
OF  THE  STARCH,  PROTEIN,  AND  NONPROTEIN  NITROGEN 3^ 

Exp er iment  XIII.  Does  colloidal  iron  precipi- 
tate starch?  --------  ------  39 


V.  MODIFICATIONS  IN  TEE  VAN  SLYKE  METHOD 


40 


-iii- 

page 

VI.  THE  iviSTHOD 

IN  DETAIL  

U1 

PART  I. 

Preparation  of  the  Sample  ------------- 

Ul 

PART  II. 

Prepara-tion  of  the  Hydrolyzed  Protein  Solution  - - 

41 

A. 

Extraction  with  anhydrous  ether  ---------- 

41 

B. 

Extraction  with  cold  absolute  alcohol  ------- 

4H 

c. 

Extraction  with  cold  1.0  per  cent,  trichloracetic 
acid  ---------------------- 

^3 

D. 

Extraction  with  dilute  sodium  hydroxide  ------ 

43 

E. 

Digestion  with  h.C  per  cent,  trichloracetic  acid  - - 

44 

F. 

Extraction  with  20  per  cent,  hydrochloric  acid  - - - 

44 

G • 

Extraction  with  strong  sodium  hydroxide  ------ 

45 

H. 

Treatment  of  the  cold  1 per  cent,  trichloracetic 
acid  extracts  ------------------ 

46 

I. 

Treatment  of  the  2 per  cent,  trichloracetic  acid 
extracts  -------------------- 

47 

J. 

Determirjation  of  insoluble  humin  nitrogen  - - - - - 

48 

PART  III.  Analysis  of  the  Hydrolyzed  Protein  Solution  - - - 

48 

Determination  of  ammonia  (airide  nitrogen)  - - - - - 

48 

Soluble  h-umin  nitrogen  - --  --  --  --  --  --  -- 

48 

Precipitation  and  washing  of  the  bases  ------- 

49 

Decomposition  of  the  phospho tungstic  precipitate 
by  a mixture  of  ether  and  amyl  alcohol  - - - - - 

51 

Determination  of  arginine  ------------- 

53 

Determiriation  of  the  amino  nitrogen  of  the  bases  - - 

53 

Determination  of  the  total  nitrogen  of  the  bases  - - 

54 

Determination  of  cystine  ------------- 

54 

Determination  of  the  total  nitrogen  in  the  filtrate 
from  the  bases  ----------------- 

55 

Determination  of  the  amino  nitrogen  in  the  filtrate 
from  the  bases  ----------------- 

5b 

-IV- 

page 

Calculation  of  arginine,  cystine,  histidine,  lysine, 
and  the  non-amino  nitrogen  - --  --  --  --  --  - 

(a)  Arginine  nitrogen  ------------- 

(b)  Cystine  nitrogen  - --  --  --  --  --  --  57 

(c>  Histidine  nitrogen  ------------  57 

(d)  Lysine  nitrogen  --------------  57 

(3)  IJon-amino  nitrogen  in  the  filtrate  from 

the  bases  ---------------  57 

Purity  of  reagents  5^ 

VII.  DIITERI.'IIHATION  OF  THE  AIvlIITO-ACIDS  OF  OATS,  CORIJ,  COTTOHSEED  MEAL, 

Al-ro  ALFALFA 59 

TABLE  I.  Distribution  of  the  nitrogen  of  oats,  corn,  cot- 
tonseed meal,  and  alfalfa.  (Results  expressed 
in  percentage  of  the  total  rh  trogen  of  the 
feed^  ---------------------  ol 

TABLE  II.  Distribution  of  the  nitrogen  of  oats,  corn, 
cottonseed  meal,  and  alfalfa.  (Results  ex- 
pressed in  percentage  of  the  feed)  ------  6c; 

TABLE  III.  Comparison  of  results  v/ith  those  of  previous 

investigators  ---------------  06 

TABLE  IV.  Nitrogen  distribution  of  alfalfa  --------  71 

VIII.  SUMMARY  AND  CONCLUSIONS 72 

IX.  BIBLIOGRAPHY  73 


THE  DEVELOPMEl^T  OF  A I.IETHOD  FOR  THE  DETERIVIINATION 


OF  THE  AMINO-ACIDS  OF  FEEDS 
I.  INTRODUCTION 

Investigations  of  the  past  few  years  have  shov/n  very  clearly  that  the 
total  nitrogen  determination  and  subsequent  calculation  of  crude  protein  there- 
from, alone,  as  has  been  dona  in  the  past  and  is  still  being  done  at  present  to 
a large  extent  in  feeding  and  metabolism  experiments,  is  of  practically  no  value 
for  the  purpose  of  estimating  the  true  nutritive  value  of  the  proteins  of  a feed. 
Before  the  true  nutritive  value  of  the  protein  content  of  a feed  can  be  accurate- 
ly determined,  among  other  things  the  amounts  of  each  of  the  various  amino-acids 
(or  at  least  certain  ones)  v/hich  go  to  make  up  the  proteins  present  must  be 
Inovm. 

Notwithstanding  the  many  almost  unsurmountable  difficulties  connected 
with  the  isolation  and  purification  of  na-turally  occurring  proteins,  a very  large 
number  of  vegetable  and  animal  proteins  have  been  isolated,  purified,  and  the 
amino-acids  present  determined  by  methods  available  for  that  piurpose.  The  vari- 
ety, the  extremely  complex  character,  and  the  ease  with  which  proteins  are 
char^ged,  make  a general  method  for  the  quantitative  isolation  and  purification 
of  all  the  proteins  of  a feed  impossible.  Osborne  (1),  in  his  Monograph  on  the 
vegetable  proteins,  states,  »'The  solvents  used  to  extract  the  proteins  of  seeds 
are  water,  neutral  saline  solutions,  ]0  to  SO  per  cent,  alcohol,  and  very  dilute 
acids  and  alkalies.  By  the  successive  application  of  these  solvents  the  greater 
part  of  the  protein  can  be  extracted  from  most  seeds  when  finely  ground,  but  the 
residue,  even  after  extracting  as  completely  as  possible,  usuftlly  contains  more 
or  less  nitrogen."  Osborne  and  Mendel  (2)  found  approximately  6 per  cent,  of 
the  total  nitrogen  of  whole  corn  left  after  extractions  with  10  per  cent,  potas- 


*•> 


^ , t 


'< 


\ 


■m 


-•5. 


cr.ij  ' ‘ '. . . 


- • ■ T. 


• 1 i-  ^ •' 


.-r,>  ^.:.  If’  •fiOi  ‘ 

-<'•  -;  ,;jl  Unx  i:  - '■■  .’^-t  Cl  ri 


*r  •}-;■*  ■ 


jl»V-  J * '!  t ^ -uOiiP , ..:co*ii 


. • ir’,^.’.--, 


.'•  i-' 


JW  ' - - .’ 


.•.-T. : £ - a *■  Lp»*  *■  *■  - 


:’... i 


j3 


,^'.1'^  ♦o-.  , •*  7^- ■ oy 


f -3'yt'u  jiiisi  vA  *i<5) 


•wOfti 


trit/!: 


t:..£ 


a ^ i-a  ■ 


*.  «a;  ;,.  r.-iJ  ‘>10«  ■ 

K :u  . , • :'  ■>rfi&j.5'-xaX*3t'^P  '■^  ’ 

.,  . •.•'  . -..•  . . j-‘:*«Ov.)’i  A *10  IH-.ivfnVi  «a:‘  ^1-  ''^• 

•■  ' .j  ' **  ^ H *.  ^.  .■>■••  ■•  " - _i 

»ui  5 = ^'---x:  r -I  ■■  •i®»30v 

^ ^ # ..  ^ ^ - -r''  . fwv^  ^ 

f i.  »4*  w:  » *■-  j y ^ ^ 

. - ^ . er  r*  ' « J J €■  i-'  Y ^ 

'I  ••;> .•a;A».fli  - ' V.-- ;:■  1-1  - -U-  • ■■’  ■■'' ■>■»'<■ 

,.:--J  - ...^  . ..  »*  ■-Si'*..  • -■■■  :■•>=•-:-•  •■«-.■ 

..-  ,,H.  : ::-.■  UU’SI  (S)  frfysMl  ” •’••  ''  • - tAi  rr 

- . ••  -x'-.  v l .'.-JVI  i.-!Qi-*';i.-t!i;s  3>-  -i'J  ■>  ■>« 

a.-  -'9Bae0aaMaqp««MaY*^ 


I 


■';r 


r ;'  ^‘5  *1“  . 


sivm  chloride  solution^  90  psr  cent,  alcohol,  and  0.2  per  cent,  potassixm  hy- 
droxide solution.  Miller  (3),  in  a study  of  the  distribution  of  nitrogen  in  the 
alfalfa  seed,  makes  a 0,5  par  cent,  potassium  hydroxide  extraction  and  leaves  be- 
tween  9 si^nd  10  per  cent,  of  the  total  nitrogen  in  the  residue.  Dowell  and  Menaul 
(4)  report  9<i  per  cent,  of  the  nitrogen  compounds  of  pecan  flour  (100  mesh)  was 
soluble  in  0.2  per  cent,  sodium  hydroxide;  ]0  per  cent,  of  the  total  nitrogen  of 
peanuts  was  soluble  in  5 per  cent,  barium  hydroxide  while  9^  Per  cent.  v?as  soluble 
in  0,2  per  cent,  sodium  hydroxide;  62  per  cent,  of  alfalfa  (40  mesh)  was  extract- 
ed by  a 0.3  per  cent,  sodium  hydroxide  solution;  and  the  mximum  extraction  of 
nitrogen  from  kafir  (60  mesh)  was  36  per  cent,  of  the  total  nitrogen  of  the  san^l<  . 
It  is  certainly  very  doubtful  whether  extractions,  which  leave  so  much  of  the 
total  nitrogen  unextracted  in  the  residues,  contain  representative  protein  mater- 
ial of  the  sample. 

The  excellent  investigations  of  Fischer,  Hausmann,  Kossel,  Yan  Slyke, 
Dakin,  and  others  have  made  it  possible  to  determine  the  various  amino-acids 
making  up  the  protein  molecule  of  pure  proteins.  But  it  is  at  present  impossible 
to  calculate  the  amino-acid  content  of  feeds  from  the  amino-acid  content  of  the 
isolated  proteins,  first,  because  all  of  the  proteins  of  no  single  food  or  feed, 
with  the  possible  exception  of  milk,  have  been  isolated  completely  and  in  a pure 
state;  and  second,  because  as  yet  there  are  no  metliods  available  for  the  quanti- 
tative separation  and  purification  of  all  the  proteins  of  a food  or  feed. 

II.  HISTORICAL. 

Grindley,  Joseph,  and  Slater  (5)  made  the  first  attempt  to  determine 
the  amino-acid  content  of  feeds  directly.  Tlieir  method  consisted  simply  in 
complete  hydrolysis  of  a finely  ground  sample  of  feed  with  20  per  cant,  hydro- 
chloric acid  and  the  application  of  the  origi:ml  Van  Slyke  Method  (6)  to  this 
hydrolysate.  The  results  v/ere  expressed  as  percentage  of  the  total  nitrogen  of 


-3- 


the  faad  and  as  percentage  of  the  feed.  Soon  after  the  above  paper  appeared, 
Nollau  (7)  published  results  on  »' the  axnino-acid  content  of  certain  commercial 
feedingstuff s and  other  sources  of  protein.”  He  also  used  the  Van  Slyke  method 
which  other  investigators  had  used  so  successfully  in  the  determina,tion  of  the 
chemical  groups  characteristic  of  the  different  amino-acids  of  proteins.  The 
procedure  used  was  as  follows:  The  samples,  ViThich  were  ground  so  as  to  pass 

through  a 40  mesh  sieve,  were  extracted  with  ether  to  remove  the  fat.  Tns  sam- 
ples were  then  completely  hydrolyzed  by  boiling  with  dO  per  cent,  hydrochloric 
acid,  the  insoluble  residue  filtered  off,  and  the  clear  filtra,te  analyzed  accord- 
ing to  the  Van  Slyke  method.  The  results  were  calculated  on  the  basis  of  the 
total  nitrogen  in  the  filtrate  from  the  insoluble  residue  and  not  on  the  total 
nitrogen  of  the  sa.nrole  of  feed. 

Later  in  the  same  year  Grindley  and  Slater  (o),  using  the  same  method 
as  that  used  by  Grindley,  Joseph,  snd  Slater,  published  additional  results  on  the 
same  problem.  The  differences  in  the  procedures  used  may  explain  to  some  extent 
the  lack  of  concordant  results  between  those  obtained  by  Hollau  and  those  obtain- 
ed by  Grindley  and  associa,tes  but  the  difference  between  many  results  from  the 
two  laboratories  is  greater  than  the  differences  in  procedures  would  warrant. 

As  subsequent  criticism,  of  the  application  of  a method  designed  en- 
tirely for  the  p\irpose  of  analyzing  pure  proteins  to  the  analysis  of  heterogene- 
ous mixtures  such  as  feeds,  pointed  out,  the  effects  of  the  nonprotein  nitrogen- 
ous constituents,  the  carbohydrates,  fats,  etc.  were  unkno’wn.  In  order  to  de- 
termine the  effect  of  the  nonprotein  nitrogenous  imterial,  Grindley  and  Eckstein 
(9)  studied  the  nonprotein  nitrogenous  material  extracted  from  various  feeds 
with  cold  water.  Hart  and  Bentley  (10)  also  made  a study  of  the  "vvater-soluble 
nitrogen  of  soma  comiTion  feedings t'^offs,  ” b\it  used  hot  water  instead  of  cold  water 
as  their  extracting  fluid.  From  the  fact  that  most  of  the  nonprotein  nitrogen 
of  the  feeds  sxa.mined  was  in  the  form  of  ammonia  or  oC-amino -acids,  free  or  com- 


. ^ r.t^sri  .i.  ‘ 4. 

* :•’  . ' i'  '■ . .4. i’ "'l 

r'i  ri'i-  ' Jv  owi  ^ 

ii^  ti.  '-’iru  -3>  5.  ■■  K' 


. - f Dili 

« < ,3  O b;.  . '.  t 


"^■s:  r:- i : :xi  c!tX4'' 


f.prc'ia 


■L'i- 1.  l:.7.r . 

. S \ ..9 'i.-- 


»• ' i . 

; 7 

/>  •• 

6*rXjbftb<i7  7 

•f  »•« 

, 1 4 

Op 

1 . ^XlL.'  ' * .»  k. 

.i ' >■ 

• u ».'i-  .k  ^ 1 *• 

- :p-C3f«I':r 

r..; 


; J- 


..‘.  “_.J?  : t .^V.12  f V 3^- 


*^<'■0- ■' *.'*7  Ixj,4c; 


■e  I 


j » '.«' 


4«‘. 


- -.. 


' } : 73*.*^-  X i»i,.'.x»a  e:'j  ..1-79?^! 


w.’%v  IT  ?■  j '. 


i 

I 


-•'■■  l *1  I . »,■  C * ; .7  -/iX  4 ^ wl  : -f  ^b 


?L->',  ,v * i •;  V J’  - 
.ri-  irfoir.  *»Jy'V. 


I 


;••  ■'  • ?uc;i'r  £-■  •'••<£-£•  riv;iT  ^.  ■'■fii.  i:  nj,; 


.iJw'i.i  iC- '“i  .iti- -i  ti'3^- 

;•  •.--  • . ^ - • ‘ -.V '-  -•  -t^  rU  ' : >-”•■ 

J .1  .,<• 

Pi:-  ,- 


- . ^ . ■'  . \t  L> 

ti  .-  ij.,.  fT«' i j x»:ii5u'-£.k.  c-'- 

..  . ."•£.■  rfs>  i 


.O. 


- ‘if  * •. 


1-'  .'^.  ’C'ii"''  r ■ . 1 

i •t'-' 


, I 


? 3:o 

i-:  . '.-.f.'r'Mic  c ■ . •«.  ^trrnAviprf-fl*.  ‘ •.-.•^c.  «4v 

Xi..'-,  ■ ,-x.*;.i.x  .7,.  i'i,u  : "Xi  ■-■ 

-V  r --7. tx*  iTtl'iv.  M*e>p  «!'0.‘''«“i0':3  • n 

i«  a,.j  . 


*.'::  ,..  ‘ "t.  7 :i.  •v:,'y> 

✓ 

7X;:,C^Jk-:  -'X"  r.  •.  .7i  - 90i  'ic- 


,xi.o  s -K  if!  'uL)  ’ 'J.xtvR  - 1- X»  r,*l'n 

•i  : ■ X,  ^r:  ,.^,.1  '-T  .:- .r-^::  c- 

'.  -j  x-^xx.;  i ■ . -’JT  3.:t'3‘0x:‘‘:rv«  7^iw 

iiX  i--v-  X 


-'p-.-  4-I 7i;  ^I'le  ' 


-4- 


bined;  i.  e.j  degradation  products  of  proteins^  Grindley  and  Eckstein  in  part 

conclude," it  seams  quits  evident  that  only  a small  part,  if  any,  of  the 

nonprotein  nitrogenous  constituents  of  foods  and  f eedingstuff s can  in  ariy  way 
interfere  with  the  application  of  the  Van  Slyke  method  for  the  determination  of 
the  chemical  groups  cliaract eristic  of  the  different  amino-acids  of  protein  to 
the  estimation  of  the  free  and  combined  amino  acids  and  amides  of  f eedingstaff s." 

Gortner  and  his  associates  (11,  Ik,  I3)  have  made  an  extensive  study 
of  the  formation  of  humin  in  the  presence  of  carbohydrates  during  acid  hydrolysis 
These  investigators  have  shovm  that  the  formation  of  humin  depends  to  a large 
extent  on  the  presence  of  carbohydrates  and  that  the  quantity  of  humin  formed  on 
hydrolysis  of  pure  proteins  is  greatly  increased  by  the  addition  of  ce.rbohydrate 
material.  From  these  and  other  investigations  the  chief  source  of  error  in  the 
method  of  aralysis  used  by  Grindley  and  associates  and  by  Nollau  was  thought  to 
be  caused  by  the  presence  of  the  carbohydrates  in  the  feeds  during  the  hydrolysis 
and  subsequent  analysis.  Grindley  (14)  makes  the  sta-tement:  "Tne  high  results 
for  humin  nitrogen  probably  constitute  the  most  serious  of  the  errors  involved 
in  the  application  of  the  Van  Slyke  method  to  the  analysis  of  feeding  stuffs, 

" And  that  no  claim  to  perfection  is  made  for  the  results  published  by 

Grindley  and  associates  is  shown  by  the  statement  of  Grindley  (14):  "Further  it 

is  also  quite  evident  that  the  results  so  far  obtained  in  this  work  are  only 
approximately  accurate  and  at  present  are  to  be  considered  of  canparative  value 
only." 

Eckstein  and  Grindley  (I5)i  in  an  attaupt  to  reduce  the  quantity  of 
humin  formed  during  hydrolj’-sis,  made  two  decided  improvements  on  the  older  method 
of  Grindley  and  associates.  The  first  was  the  removal  of  some  of  the  nonpro- 
tein nitrogenous  constituents  by  complete  extractions  with  ether  and  with  abso- 
lute alcohol.  The  second  was  the  treatment  of  the  residua,  remaining  from  the 
ether  and  alcohol  extractions,  so  that  the  greater  part  of  the  carbohydrates  was 


I ^ 


.3--. 


A-  . Ct  f 

. . j,- 


a u i. 


.ZZj 


iLffU  --i  -V  ; ■ ^3.  ' : i 

;,^--i  •-■  ..^  . ; ’io  .Vi'  - :.. 


. *1m3 


^ ' r *■ 


^ Ttu  Ji  •^'.  - 


i ~ 1 '^  1 


- * : - '-C.-  1 


. ‘ . . ..  i - ■'  -t.  ..A 

'■  r 

' , .•  - i-i-  ii  - -■•♦ 

:r.J-f  L-rr.,,  •i'^  ^ *1-  HviJ^ 

^ . i i - - , » - - • » 

^ , - . -r  T 1 .tioi/rf  Aj'-n.ri  ?-  a'  - 

•►4 

, ■■r.,  ■ ■}  ;*r  .3  <TW>.-..  * . »i  >;  “2  -■  *.fi  -C»eiii 

* . . ■• 

:;.  ■ . ■ .-j  > ■ ... ''n-*-'  •"  oa-'" 

'.  ■ •■'»;!.  '•  ...  J.ii'  -■  t-  '■  *^*'4 


1 .-  .:.'  tO  A- 

» 

fi  .A..  -,. 


;^i^v  ■ . i ." 

I 03“.,  A*-  2 


1 . % 


i 


....O  .V-  .si:....-:-  ■.  --  •/'  :iA- 

• . yf>f  ■>«.•'  • li  'jfco  '-  -i  i -1 J '^y  Lt-ts".  _ .. 

Ln^B  li'-.  c.r  i •^'•;  ij*v.  .r  v.c  - .-  .i.,  - • ..x-.i  u .-wi 


J Z>-  *■ 

J 


-a.  or-. 


- , t ' *...  •--',£  £.C..J 

,C  crXfc  .I.  •■  ^fc.  J >1  ■ j ^i-.-..'  ....I,  A 


•is  St,:*\ 


t.:.  ^*:  o;;  ■ ■ ' '■  --'-a  -.i  ^ c; 


-R- 


separated  from  the  main  portion  of  the  proteins  before  the  latter  are  hydrolyzed. 
The  details  of  tne  method,  as  given  by  Eckstein  and  Grindley  (15)>  fol- 

lows: 

•'Weighed  quantities  of  the  feedingstuff  are  extracted  with  ether  in 
Soxiilet  extractors  and  then  with  cold  absolute  alcohol  on  Buchner  furaiels.  The 
residues  thus  extracted  are  digested  for  I5  hours  three  or  four  times  vath  0,1 
per  cent,  solution  of  hydrochloric  acid  until  all  the  starch  has  been  converted 
into  sugars.  The  residues  insoluble  in  0.1  per  cent,  hydrochloric  acid  are 
boiled  with  cO  per  cent,  hydrochloric  acid  -until  the  proteins  which  they  contain 
are  completely  hydrolyzed. 


•Tne  filtrates  from  the  residues  insoluble  in  0.1  per  cent,  hydrochlor- 
ic acid  are  neutralized  with  sodixm  hydroxide,  then  faintly  acidified  with  acetic- 
acid,  allowed  to  stand  over  night,  and  then  filtered.  The  filtrates  fromi  the 
precipitated  proteins  are  concentrated  vacuo  to  small  volumes  and  precipitated 
by  the  addition  of  five  volumes  of  absolute  alcohol.  After  standing  over  night 
the  precipitated  proteins  are  removed  by  filtration  and  washed  w'ith  83  per  cent, 
alcohol . 


•'The  filtrates  from  the  proteins  precipitated  by  alcohol  are  concen- 
trated to  small  volume  and  enough  concentrated  hydrochloric  acid  is  added  to  make 
a 5 per  cent,  solution.  The  solutions  are  then  boiled  until  hydrolysis  is  com- 
plete. The  proteins  separated  above  b3’-  neutralization  and  bj'-  the  addition  of 
alcohol  are  boiled  with  <£0  per  cent,  hydrochloric  acid  until  hydrolysis  is  com- 
plete, 

•'As  a result  of  the  above  procedure,  there  are  obtained  three  differ- 
ent fractions  of  the  proteins  of  f eedingstuffs  which  are  completely  hydrolyzed; 
namely,  (1)  the  proteins  insoluble  in  0.1  per  cent,  hydrochloric  acid;  (2)  the 
proteins  separated  by  neutralizing  the  mineral  acid  and  precipitating  with  alco- 
hol; (3)  the  soluble  proteins  not  separa.ted  by  neutralizing  the  mineral  acid  or 
precipitating  with  alcohol.  Each  of  the  three  hydrolyzed  solutions  is  filtered 
and  the  insoluble  humin  substances  are  repea.tedly  digested  with  0.1  per  cent, 
hydrochloric  acid  and  then  thoroughly  washed  with  hot  water.  The  nitrogen  in 
these  residues  is  considered  to  represent  the  insoluble  h\xnin  substances. 

"The  filtrates  from  the  insoluble  humin  were  concentrated  in  vacuo 
to  small  volume  and  the  ainide  and  soluble  melanine  nitrogen  determined  as  usual. 
The  remaining  parts  of  the  analysis  are  continued  as  usual." 


This  method  gave  the  followii-)g  results  for  hur.in  nitrogen,  as  percent- 
e.ges  of  total  nitrogen  of  the  feeds:  corn  3.2,  wheat  oats  barley 

3*9  psr  cent.  These  results  maj’-  be  compared  with  the  humin  nitrogen  of  the  same 
feeds  by  the  earlier  method  of  Grindley  and  associates.  The  humin  nitrogen  val- 
ues then  were  corn  Q.8,  wheat  9.2,  oats  9«9>  barley  8.8  per  cent,  of  the 


total  nitrogen. 


■■-An  . 


■ . '1  . ■:  .-Z^;  O'^H 


4 


-'.o'-.  .V 


,f)0iijttai  * 1®  sTi  ' lb  T 


i ,:.i'  A'  Z*  Z />6£.  Zv?t''V  ; 


'Jlt.'vj  l~..' 


^ V ^ j il 

•. ; iw.-.  : 

: -r.-:  . -.lAr^aU- - ^ 


'■  f ■< 


:*a 

A " .-«.•••* 

.*r ; :''•  - ,*t 


.■‘*:t’\jz  :o  it:J*Q9  . 'ics. 


^ '9 c>'_T  . " j 

■ : T*q  ^ ..J*.V  i 

■ ’iZTij/i  . . .'  c ~ j. , ;"  I I.'- 


.^«k! 


».  * v*^  ' * 


-';■  V.  • .T : U-»i3 -■ 


'ivv 

J « ' 


-iC":  k ^ ®-‘-  _. 

■ *;.  u;.£..'  ’ *X.-.  !?■':»&  O- 

' .’  rw  ' (J-*  * ” *»c  * » 1"*' 

. ! •*  \'.Az".iso  »■;/  ■ J4C  fqio«i  i:; 

■"'v'Toe^f.  . . «*rw.?r  vX^’  fi^A: %;o 

.,*j  ie<  • r ••  .r  -X»  ..jZc'C'-i.  •••cwr  .i:ai-l 

' "*'i  ■ '■ 


« ; it  . r. 


,'  :ii.  tj-Z'T.  ' •HIO"  : 

X - . .-  i "'X  ^ V r . . iX  ■ ^ 

.-I  ; ••  ->  j,  j . -"I  ^ 


..  : :iri  ■ 


'V® 
- *" 


Iw 

•#»  ■■"‘i 


. i - t>,  > , 

.^V  1.^.  . ■’  -->5v 

' _,Icr  . "o 

■•  . ''i'. 


4**^i*j  rf  • 

if*  ■->  - 


ev^xfR.  > . }-..'i:ri  • 


'1 


1 


..... 

• •-  :i:v;  1 

r *_»  j,' 

i'..'  :.  . ■.•:>  i«  •-c'..  ....  ' '' 

--H  • - ■ - *•  - ■ ' •'■'  • ’.  • ' ‘‘  " ^ Z -••:4.  jv  ■ 2.:  X®i  ■’•! :, 

'■-.  f -J-  . - ..  Cl^..  ....  ' . , \ 'f 

' ■ -iH‘.  r vf'  V - ura-  . * ;V-  ■V.-.'wV,  c- ' ^■„;  i • >» 

;o  .iO..  ••  . '-r’oox*  .•-ilvr>'*l  ■ 

,.  ^ . < Xi  < . 'A.  ..•':  . e " *•  '•^^...- t'Xdtfi 

, V-'  - ’^v'  -2;r  H --  . Z •.  j Z ..DOib'tr. 

;;  '>”'r  4*-::  ^ r ti  '* 


a? 


■ .j.' i.  ■ . u.w 


. - -*'i  *='  S ' 


■;  Ufj  2'-  ■*.  3 r 

. / ♦ u . ■ 

MW  . . 


i.ftT'  VN. 


-li.-J'v'.  ■ •fc’’  • 


: . 


Lj 


*1.  '’-i 


. 1 

•; . 


■ ..'C  2 7 t tiU* 

>,  ' rr;  ■••‘-  at  t©iTi : 


*JO  5^1-i  . _/  OS'2 

•76,23  tif.i 

.•3  -10 5 ^ 

• y±90\ 

.-'>.-'3  tMT 

.■  3.  ,ri 


aay^at^MTcai 


-6- 


The  highest  hiamin  nitrogen  reported  by  Van  Slyke  (6)  for  pure  pro- 
teins was  3.6  per  cent,  in  the  case  of  ox  heraoglobin.  Hartley  (16)  reported 
2.5  per  cent,  humin  nitrogen  in  euglobin  of  ox  serum.  Van  Slyke  (6)  found  7*4 
per  cent,  of  humin  nitrogen  in  dog's  iiair.  Thus  the  quantities  of  humin  nitro- 
gen obtained  by  Eckstein  and  Grindley  compared  very  favorably  with  that  found  in 
the  analysis  of  some  of  the  pure  proteins. 

III.  DEVEL0PI:IEITT  OF  THE  METHOD. 

While  the  new  method  of  Eckstein  and  Grindley  decidedly  lov/ered  the 
quantities  of  humin  nitrogen,  especially  in  the  arialysis  of  cereals,  several 
difficulties  were  also  introduced.  Possibly  the  most  serious  of  the  difficul- 
ties encountered  in  the  use  of  this  method  was  the  fomation  of  a dark  colored 
substance  during:  the  precipitation  of  the  bases  with  phosphotungstic  acid.  Some- 
times this  de.rk  colored  substance  was  of  a sticky  nature  and,  \>hen  present,  made 
thorough  washing  and  a clean  decomposition  of  the  basic  phospho tungstates  impos- 
sible. Neidig  and  Snyder  (I7)  encountered  the  same  difficulty  in  applying  the 
method  of  Eckstein  and  Grindley  to  silage  crops.  They  refer  to  this  fraction  of 
humin  as  the  "phosphotungstic  humin"  of  Gortner  and  Holm.  This  fraction  of  humin 
nitrogen  will  be  referred  to  again  later.  It  has  been  found  (see  Experiment 
XII,  page  33)  that  5 psr  cent,  hydrochloric  acid  very  probably  does  not  ca.use 
complete  hydrolysis  of  the  proteins  soluble  in  the  hot  0.1  per  cent,  hydrochloric 
acid  and  are  not  precipitated  by  either  neutralization  or  5 volumes  of  alcohol, 
by  boiling  for  "only  a short  time."  (From  unpublished  papers  of  Eckstein  and 
Grindley  this  time  was  6 hours).  It  was  also  found,  by  the  use  of  the  Eckstein 
a,nd  Grindlej’’  method,  that  the  total  nitrogen  finally  recovered  was  alva,ys  some- 
what less  than  100  par  cent.  As  was  pointed  out  by  Neidig  and  Snyder  (17)^  the 
per  cent,  humin  nitrogen  obtained  in  the  analysis  of  forage  crops  by  the  new 
method  of  Eckstein  and  Grindley  v;as  only  slightly  lower  than  that  obtained  in 


't  • i.-- 

) •'♦I  , -t"'  1- 

m i-  ' . - 1 ^ V 


. ■ .,  aaiei 

1 .'  Llar‘ 

i.z^u:  . tr.^o  "i. 


i*q 


.;..v,''  yv^c  .._:-  l>:3i rt  n * ‘ > j t I vri  isitlAfS^  cii>r^ 


. *-i.:-»'  -*'X  1,  f<“.  , 


c ;r  \o  q^t^rAn/?  * ■ 


■.:  ;rj«c  i,  tj5  ’icl  io£.:tii  woa  •_ '.‘ 

,^:'  ' C .-i  ’ ^ • -*■*  fu-'.’ri-  H9lCli:'.'~- 

.,  :•.  . -i  . . . ••  ‘ - lE't'  ’-‘I ; ■ * 

...  '-.•  . '■iU  J />H  >rt!  JJ'  0 ir  jC-tol?  ■ i”.-  ' f b9i 

^:::-  •■  -.,- _ . „•' c;/  ' c'  ; . ica  I *i iu”"  V-  -*  n/'at.  ■ 

. . .•  -;.  - -'<w  i;  -at-  "«*:oIc5  aC*!%L  ai*iJ  i:.ri 

; ...j,*i  f ,..■  '!■'  .,  "r.  .'.*£■%>--•— 'i»&b  ~.X»fIc  ’ J r' : •JiXiOUJn 

.:  -.■  f'il' 


«.  S.t/i 


0 3 1 ■‘*-  . 

6^ii- 

V ■?  i r.vv;": 


•:*.i.Jf.  *c  . '^inu  ■ 


V-'l  ’ ■X9V  tit*’  1.-.  v*lt' 


' {. . i %.t 

r1  ij  * 

«d,  i|T"  * 

V.-:  ^ .S-: 


. '.*..  . , . .'  • * Xi.0  :;.-v 

. ...,,. -J.r.-.  -if JX'.'-.xi  Joaf-rt  *r.-.J)ic^ 

. - . >4  i..  . - y z.'^)  ’:*•••■.■'  :liOl  v- 

r.r.c-  .:  v-^  : y >rf  & tiw  ^-•••‘'•'  ..sXtMtitC 

i •*  *:  .•'••"  •■■  edi  Icciii 

.2  slt>2f  r '■  tf-lc*  i ..  iO  •:*-£  uvx  '-x,.:. 

•se^O*'  j i l*;  , * — f 


-7- 


the  analysis  of  the  same  feeds  by  the  original  method  of  Grindley  and  associates. 
It  was  not  these  objections  to  the  new  method  of  Eckstein  and  Grindley,  alone, 
which  lead  to  the  search  for  a better  method.  In  all  the  previous  methods  for 
the  application  of  the  Van  Slyke  method  to  the  analysis  of  feeds  the  free  amino- 
acids  e,nd  amides  as  well  as  the  combined  aa'.ino -acids  and  aiiiides  were  determined. 
That  is,  the  free  anmonia,  free  amino-acids,  and  amides,  vdiich  constitute  the 
main  portion  of  the  nonprotein  nitrogenous  material  of  the  feed,  were  determined 
along  with  the  combined  amino-acids  and  amides,  which  represent  the  protein 
content  of  the  feed.  A method  by  which  the  protein  and  nonprotein  nitrogen 
could  be  separated  and  at  the  same  time  the  distribution  of  each  determined,  if 
desired,  v;as  highly  desirable. 

In  the  sea-rch  for  a better  method  a la,rge  number  of  separate  experi- 
ments were  carried  out.  The  experiments  varied  from  only  slight  changes  in 
technique  or  slight  change  in  reagents  used  to  almost  complete  revision  of  the 
method  previously  used,  ffeny  tests  gave  negative  results  and  many  gave  positive 
results  but  for  some  reason  could  not  be  used.  Only  a fev;  of  the  more  important 
experiments  which  had  direct  bearing  on  the  development  of  the  method  will  be 
recorded. 

A.  EXTRACTION  OF  THE  NONPROTEIN  NITROGEN. 

(1)  Extraction  with  anhydrous  ether. 

Nollau  (7),  in  his  application  of  the  Van  Slyke  method  to  the  ar.aly- 
sis  of  certain  feeds,  extracted  the  feed  with  ether  to  remove  the  fat.  Eckstein 
and  Grindley  (IE)  made  ether  and  alcohol  extractions  to  remove  some  of  the  non- 
protein nitrogenoiis  constituents  such  as  the  nitrogenous  lipoids,  coloring 
uiatters,  etc.  In  the  method  finally  adopted  for  this  v/ork  both  anhydrous  ether 
and  cold  absolute  alcohol  extractions  are  made,  but  the  method  of  carrying  out 
these  extractions  is  somewhat  different.  Instead  of  the  ether  extraction  in 


- :r  I'-.  «m*«  i.  J ■ ..'  mli 

,r.v:  i. ; . Ii-.i'!  \v  '^.i:  is?  •:  ■ fJfd’  *'"  '*  J-  !'] 

;■:.  . . V '•  ■ ..  . .-.-r  »:«.!•  ''s'.  • - ^ ^-..'1 

- •;'■  - '•  j -l-  ~ -■-  >v  'r?  *c:j.  t ’ ; ' 

. le,  ?•  <'*  L-1  > t fjc;  : T :!“'■■  ‘ i ^,%S  i*  || 

• ’ ' — iri-ir  i-Vy  l"^•  '.'ll  .S*_  .’•'il 

C5-'  ■ 

f»i»»vr  ,li»i%if-.i'  .:  ..  - .V  . _ v':-  .•-»■ ..T  xii«  ■ oft-  Is.’  r.l . , j 

.':  I 

•Vi 

nl'-.:  •:'  ' • . : '..■  ■ ■■;♦••:  i ^ l.  i«  t»r- ' 1 -..  *iJi3'-.’  or  ftJir’  gftvl  *' 

\ 

i 

\ 1 

■ .oi  - -ij  • .'  '■  . A v‘  , ed  hi',:--.  i 

. 1 >u-?;  r.  • 

•'  •.  ••■;■•■:  V'-  ’•:  (.  .»*  *■  5:.'.  .•'  ir-jv:  « -^o  ' ■,'.  il 

i ^ ..  ..  . i-  ».  - r'.:  : e. -i.-V  li  -^rx»  %:<1  . ^ 

r ■' 

>:•  vr-  r • ■• , ,.‘c  ..  -\  .•-.  . tf  ’ •■  '*■  '"k-  *"'  • ^ ti 

i- 

V ...  -“'v  , 1 'i-'\.  ■■  • ■ r-- j - K . ^o■'4e•; 

Ti  ..-  J vC'V  ■»  '*  I '•  v.-r  : , ■ j.'  ^ ..  ■ - ■ ■ .vfaAOl  ft  - . . "TOl:  v .vf  SJli'iCT  ^ 

«■:.■  -Hv  r-..;;,: -.  r , V'.{  • ■■  ■ 'xr^  * *■ 


-3- 

Soxiilet  extractors  it  lias  been  found  much  more  convenient  to  UiSdce  the  extrac- 
tions of  the  sample  in  a centrifuge  bottle.  This  is  the  method  adopted  for  all 
extractions  in  the  cold  and  in  general  is  carried  out  as  follows;  The  sample  is 
placed  in  a 5OO  cc.  centrifuge  bottle  and  100  to  200  cc.  of  the  extracting  liquid 
added.  The  bottle  is  corked  securely  and  placed  on  a mechanical  shaker  which 
keeps  the  bottle  and  contents  in  continuous  motion.  Usuadly  but  tv/o  extractions 
are  made  each  24  hours;  one  extraction  for  a,  7 to  S hour  period  is  mde  during 
the  -day  and  a second  extraction  for  a l4  to  I5  hour  period  is  made  during  the 
night.  As  a rule  6 or  7 extractions  with  a solvent  are  necessary  to  insure  com- 
plete extractions  in  the  cold.  After  each  extraction  period  the  sides  of  the 
bottle  are  washed  down  with  a few  cc.  of  tiie  solvent  or  ammonia-free  water  in 
case  of  aqueous  solvents  and  the  solution  centrifuged  until  the  solid  matter 
forms  a compact  mass  in  the  bottom  of  the  bottle.  The  supernatant  liquid  is  then 
decanted.  This  is  the  method  referred  to  in  the  follov/ing  pages  as  the  centrifug* 
bottle  method  of  extraction. 

The  completeness  of  extraction  with  ether  by  this  method  is  shown  by 
the  following  experin:ient. 

EXPERIIvIENT  I. 

Four  30  gm,  samples  of  alfalfa  which  had  been  gro\md  to  pass  through 
a bO  mesh  sieve  v/ere  ^ch  extracted  7 times  with  100  cc.  portions  of  anhydrous 
ether  in  a 5OO  cc.  centrifioge  bottle  in  the  usual  mamer.  The  ether  extracts 
were  filtered  to  remove  ar.y  solid  material  that  may  have  been  decanted  into  them, 
the  ether  recovered  by  distillation,  and  the  total  nitrogen  determined.  The  ni- 
trogen extracted  by  ether,  as  percentage  of  the  total  nitrogen  of  the  sample, 
was  0.490  cent.,  0.49«^  psr  cent.,  0,472  per  cent.,  and  0.433  cent.  An 
eighth  extraction  v/ith  100  cc.  portions  of  ether  v/as  made  and  the  total  nitrogen 
extracted  was  determined.  Five  cc.  of  N/10  acid  were  used  in  the  receiving 


■■"I 


. Mf: 


i» 

A ai 


tl  111 

I* 

- 


‘.‘•:--:c:y  e/ 


VtOKat’  9 7 


'J-  -.  .:j  ' "U 


*<«  O A . 


oj  fy^  > 


. . j«-: : -?5  1-5  ' ^ OcJ 


. f>...JC 


;y.u  "i  . ' • 


, • • '->-*0^.  >-Uw 


li;?  ' ra«X? 


i‘  '■^ 


•;5-> 


' J^'i:-v;. 

• •.  - 


inin'i'  <W>  ■ --  •• 

• . - oox  ■ »-  jfft  • 1 ■ •-■-  '•  ■ -'  • '•’ 

.' t!..*  C,  f,  tll~  i C >i.' 1-i"  >* '•' 


, ..  . _ .J  .•  .'AT.  jbiioft  cv  lO*-:  O-  ''i  .Jii'l 


4^ 


^.■ai V i t 


_o_ 


’ 

bottles  and  the  following  amounts  of  N/10  alkali  were  required  for  neutraliza- 
tion; 4.70  cc.,  4.73  cc.,  4.90  cc.,  and  4»70  cc.  The  reagent  factor  was  O.H5 
cc.  of  IT/iO  alkali. 

Since  oats  is  the  only  other  feed  examined  which  had  as  large  a per- 
centage of  nitrogen  extracted  by  either^  this  experiment  was  considered  sufficient 
proof  that  seven  100  cc.  extractions  completely  extracted  the  ether  soluble  ni- 
trogen in  the  other  feeds  examined. 

(2)  Extraction  with  cold  absolute  alcohol. 

By  carrying  out  the  alcohol  extractions  also  in  the  centrifuge 
bottle  a more  complete  extraction  is  obtained  and  there  is  also  the  additior.al 
advantage  ti^t  the  residue  is  not  iiandled  between  the  two  extractions.  In  the 
following  experiiLent  the  nitrogen  extracted  by  alcohol  according  to  the  Eckstein 
and  Grindley  method  on  Buchner  furjcels  is  compared  with  that  extracted  by  the 
centrifuge  bottle  method. 

EXPEHIlViENT  II. 

The  four  residues  remaining  after  the  ether  extraction  in  Experi- 
ment I were  extracted  7 times  in  centrifuge  bottles  with  200  cc.  portions  of  ab- 
solute alcohol.  The  alcohol  was  recovered  by  distillation  and  total  nitrogen 
determined  in  the  extract  residues.  As  percentage  of  total  nitrogen  of  the 
sample  k.Ooh  per  cent.,  2,0'i1  per  cent.,  1,809  psr  cent.,  and  2.12k  per  cent,  of 
nitrogen  were  extracted.  An  eighth  extraction  with  200  cc.  portions  of  absolute 
alcohol  contained  no  nitrogen  as  shown  by  the  fact  that  5 cc.  portions  of  H/lO 
acid  were  plaxed  in  the  receiving  bottles  and  after  distillation  it  required 
4.75  cc.,  4.7*^  cc.,  4.7s  cc.,  and  4.7^  cc.  of  N/lO  alkali  for  ne'atralization.  Thi 
reagent  factor  was  O.25  cc.  of  N/IO  e.lkali. 

Two  more  3O  portions  of  the  same  sample  of  alfalfa  were  extracted 


r 


i 


o'* 


.A 


\V. 


=i 


..  o.''.“  «i 

At^  ' 

-.J  , ■, 

n:  v.t:  u . 


: Ic 
;“V?i  fiili?  - 


C -i&z  --.  ; ■■'  ^ -*  i7*  iP‘ ; 

■ • & 


i«  cii‘ 


i.*J- 


\ 


•AH. 


.*«  ■:-• 


Xi. 


'6lr.  * ,:  ' 4 ' - 


' ? ■ r;^  5 .'  . ■’- 


.jic  r.r  .4  ' 11  jCi  '1^ 


,'i<.e>:^*>c  ^ ^CT  <- •'  •jf:  lo^T  •.'■*  •*  i> 


r j.  9:  ~ 


.-.■.:.i*-  ‘ i .;ai/S':7V.«  r-  - J 44  n:<.’  :f.y 

vie.--:-'.  ..U  !>C  ■ I..  ■ 


r>  . -2. 


At.  i • 


.1:.''  ■-  1; 


a- J:  : -£7^ » '*-t; 

. ' 0;'  ,\’.‘  t . 1 5 j u '■  £’»  ' i 

. C - » . 4 » ;.  V 5^  . . >,«J  S'  - 1 ' - 


IV- ■ - 4 ifl,  O i iO  .iC 

. *it  '.  w > 4 


v'■..^v  '!•:  {-. 


. r I . w . 


Jf  ..  e.  ■ .TVOai;  3>‘i  ix6:;oi3ic. 

r -n  'Aa  81; is 7c-''  i.-'iv.£; 

, . : . ' V 


■•  I 


m oSUMUm  lo  ai 


’i\r:  ;c  . 
. . cT.vijm  . 


‘ 4 .-.i-iiS-  -'-A 


t 


-10- 


with  ether  as  in  Experiment  then  the  residues  transferred  to  Buchner  funr.els 
and  extracted  with  1400  cc.  of  absolute  alcohol  by  the  method  of  Eckstein  and 
Grindley.  The  total  nitrogen  extracted  in  these  two  cases  was  0.4<^4  per  cent, 
and  0.646  per  cent.  Comparing  these  values  with  those  obtained  in  the  centrifuge 
bottle  method  it  is  evident  that  the  extraction  in  the  centrifuge  bottle  extracts 
considerably  mors  nitrogen  than  is  extracted  by  the  same  volume  of  solvent  on 
Bucliner  funnels.  Since  alfalfa  contains  more  alcohol  soluble  nitrogen  than  any 
other  feed  exainined,  seven  <200  cc.  extractions  were  considered  sufficient  to  ex- 
tract all  the  alcohol  soluble  nitrogen  from  the  other  feeds  if  ground  to  the  same 
degree  of  fineness. 

(3)  Extraction  with  cold  1.0  per  cent,  trichloracetic  acid. 

Hart  and  Bentley  (10)  studied  the  ‘'character  of  the  water-soluble  ni- 
trogen of  some  coiumon  f eedingstuffs."  In  their  method  the  air  dried  samples  were 
extracted  with  hot  water.  Grindley  and  Eckstein  (9)  irads  a studj’’  of  "the  nonpro- 
tein nitrogenous  constituents  of  f eedingstuff s»  and  used  cold  water  as  the  extract- 
ing liquid.  It  is  apparent  from  the  results  of  these  investigations  tliat  the  ni- 
trogen extracted  by  hot  or  cold  water  is  largely  in  the  form  of  hj/’drolytic  pro- 
ducts of  proteins. 

The  finding  of  a reagent  which  would  extract  all  the  nonprotein  ni- 
trogenous constituents  of  a feed  without  altering  the  proteins  present  or  extract- 
ing considerable  protein  material  and  the  quantitative  separation  of  the  proteins 
extracted  from  the  nonprotein  nitrogenous  material  has  bean  the  most  difficult  of 
all  the  problems  encountered  in  connection  v/itli  this  v/ork. 

Some  of  the  various  extracting  liquids  and  the  conditions  under  which 
they  were  used  are  as  follows:  (1)  cold  water  on  Buchner  funnels;  (2)  cold  0.2 
per  cent,  hydrochloric  acid  in  centrifuge  bottles  and  a I5.OO  cc.  0.2  per  cent, 
hydrochloric  acid  extraction  on  a Buchner  funnel;  (3)  extraction  with  water  and 


-11- 


0.0^  per  cent,  hydrochloric  acid  in  centrifuge  bottles^  in  which  tv/o  cc. 
amiionia-free  water  extractions  were  made  for  I5  to  eiO  hours  each,  follov/ed  by  tv/o 
H5O  cc.  0,0'd  per  cent,  hydrochloric  acid  extractions  for  2 hour  periods,  followed 
by  tv/o  more  2^0  cc.  water  extractions  for  I5  to  20  hour  periods;  (U)  extraction 
with  aim-onia-free  waiter  and  O.Oci  per  cent,  hydrochloric  acid  as  described  in  (3) 
except  that  the  acid  extractions  were  continued  for  I5  to  20  hour  periods;  and 
finally  (5)  seven  200  cc.  extractions  with  1.0  per  cent,  trichloracetic  acid  using 
the  usual  centrifuge  bottle  method. 

Water  as  an  extra.cting  liquid  for  nonprotein  nitrogen  was  used  both 
by  Hart  and  Bentley  (10)  and  by  Grindley  and  Eckstein  (9).  Water  is  a very  good 
solvent  for  the  nonprotein  nitrogen  constituents  of  finely  ground  feeds,  especi- 
ally if  the  sample  has  been  previously  extracted  with  ether  and  alcohol.  Even 
when  used  after  ether  and  alcohol  extractions,  water  extracts  the  nonprotein  ni- 
trogen rather  slov/ly  a,nd  incompletely.  It  also  extracts,  in  some  cases,  consid- 
erable protein  which  must  be  removed.  Water  very  slightly  acidified  with  mineral 
acid  extracts  the  nonprotein  nitrogen  more  quickly  and  con^letely  than  plain 
water  but  mors  protein  nitrogen  is  usually  extracted  also.  This  is  a rather 
serious  objection  for  the  quantitative  precipitation  of  the  proteins  from  the 
nonprotein  nitrogenous  constituents  has  been  found  to  be  a very  difficult  task 
in  this  work.  There  is  also  the  possibility  of  slight  hydrolysis  or  claange  of 
tile  proteins  when  treated  with  mineral  acids.  It  was  found  that,  whatever  cold 
extraction  liquid  used,  6 ot  7 repeated  extractions  in  the  centrifuge  bottle  gave 
a more  complete  extraction  than  was  extracted  by  a corresponding  volume  of  sol- 
vent on  Buchner  funnels, 

V/ater  and  0.02  per  cent,  hydrochloric  acid  may  be  used  very  successful' 
ly  for  the  extraction  of  nonprotein  nitrogen  in  the  follov/ing  manner;  Tne  sample 
is  placed  in  a centrifuge  bottle  and  extracted  twice  with  25O  cc.  portions  of 
ammonia-free  v/ater,  makirg  two  extractions  each  24  hours.  Tlie  residue  is  then 


3 .Ti  : 1 


. « 'O. . ' ■ ^ 


i^-'.  . ^ ’c- 

u.  «~oop»^ 

n — ^ ^ aCi ftiS  ^ '■ 


• ^ . i i*  ♦ 

‘ I-fci  .u?,  Ar’. 

•-;  J J..'®<I  ..:■  • :i«F  vcf 

_ •.  ;i  4-.,..‘  jC'i  ■.•'?  ■ ^ ' 

e.  1 'u 

sifTS  'I'j'ia 


; i: 

..x^rc':-* 

...,  ■..;  tlr- 

e:  . - . • 

*• 

e-^.  t-'W  :'5i:j'06lj  - 

«/J  4i,’n$&y  i --•'5I  -’4^1:1  • i’-.  r’t'ii' 

- :.  : ■ '■  3i  9 »-  • • •'■-■ 

^y-'-  ,X  i:v:&0':*  ' ■•  ; ■ 

i 'xc  c ^^9ax^  1 ^Jpif  t^oiiCvi-ii 
*rr>  riciJj^^JXS  oi^IfTiSOO  b'icr*  *■ 

. .art  ■■  ;;.*aK-  i'^'- 
r" 

Tj^'i  :)■•.  3 c‘r7;:or'  jaL*?;; '..  '‘d;  zr>t  “'•* 

*r-  ’ . ...  al  ^c'iq  J 

...f  tdfj  '-^'  . *,♦  Mf  l4‘.  T-iStffD^ 


t.vj 

. *■•*:  3;  j‘--- 

,-i 


-‘I'd- 

extracted  twice  with  cc.  portions  of  0.02  per  cent,  hydrochloric  acid,  ex- 
tracting for  2 hour  periods.  The  residue  is  then  extracted  twice  hiore  with 
£iuriOnia-free  water  as  before. 

It  was  thought  that  by  the  use  of  dil\ite  trichloracetic  acid,  since 
it  is  a comparatively  strong  acid  and  also,  in  dilute  solutions,  a good  protein 
precipitant,  the  solvent  action  of  the  acid  would  be  retained  and  at  the  se,T.e  time 
but  little  protein  would  be  extracted.  In  the  follov/ing  experiment  the  extrac- 
tions of  the  nonprotein  nitrogen  with  cold  1,0  per  cent,  trichloracetic  acid  and 
with  cold  water  and  0.02  per  cent,  hydrochloric  acid  are  compared. 

EXPERIIvIEiTT  III. 

Two  IS  gm.  samples  of  oats  which  had  been  ground  to  pass  through  an 
SO  mesh  sieve  were  extracted  v/ith  water  a-nd  0.02  per  cent,  hydrochloric  acid  in 
centrifuge  bottles.  Two  2^0  cc.  extractions  with  ammonie.-free  water  were  follov/- 
ed  by  two  25O  cc.  extractio..s  with  0.02  per  cent,  hj’-drochloric  acid,  wnicbi  in 
turn  were  follov/ed.  by  two  more  cc.  air.raonia-free  water  extractions.  Two  ex- 
tractions were  mads  each  c:4  hours. 

Two  mors  IS  gm.  samples  of  the  same  feed  were  extracted  6 times  in 
centrifuge  bottles  with  200  cc.  portions  of  1.0  per  cent,  trichloracetic  acid, 
ciakirig  two  extractions  each  24  houi's.  Each  of  the  four  extracts  were  filtered  by 
gravity  to  remove  any  solid  mterial  that  may  have  been  decanted  into  them,  made 
up  to  a definite  volvtne  and  total  nitrogen  determined  in  duplicate  portions.  The 
results,  expressed  as  percentage  of  the  total  nitrogen  of  the  feed  were; 


IT  extracted  by  cold 
wa.ter  and  0.02  per  cent.  HCl 


IT  extracted  by  cold  1 
per  cent,  trichloracetic  acid 


27.63s 

26.194 


27.731  aver.  27.0S5 
28. 333  »'  28.264 

•'  ^7.975 


14.QO7  14.629  aver.  14.76S 

14.351  14.212  ” 14.2S2 

14.525 


-13- 

The  nonprotein  nitrogen  in  this  sample  of  oats  was^  by  fo-'or  previous 
extractions  with  v;ater  and  0,02.  per  cent,  hydrochloric  acid  followed  by  precipi- 
tation with  colloidal  ferric  hydrate^  9*^^^  per  cent,  of  the  total  nitrogen  and^ 
by  eleven  previous  extractions  with  cold  1.0  per  cent,  trichloracetic  acid  fol- 
lowed by  precipitation  with  colloidal  ferric  hydrate,  10. I5  per  cent,  of  the  to- 
tal nitrogen. 

It  is  apparent  from  the  above  experiment  that  much  more  nitrogen  is 
extracted,  in  the  case  of  oats  at  least,  by  the  water  and  0.02  per  cent,  hydro- 
chloric acid  than  by  the  1.0  per  cent,  trichloracetic  acid.  Tr.at  most  if  riOt  all 
of  the  additioiml  nitrogen  extracted  is  protein  and  not  nonprotein  nitrogen  is 
shown  by  the  fact  that  by  precipitation  of  the  proteins  in  both  cases  with  col- 
loidal ferric  hydrate  practically  the  same  value  for  the  nonprctein  nitrogen  con- 
tent is  obtained. 

In  order  to  determine  the  best  strength  trichloracetic  acid  to  use 
for  the  extraction  of  the  nonprotein  nitrogen  the  following  e^^eriment  was  carriec 
out. 

EXPERIIvIEriT  IV. 

Five  30  sanples  of  oats  which  had  been  ground  so  as  to  pass 
through  a UO  mesh  sieve  were  transferred  to  ^00  cc.  centrifuge  bottles  and  ex- 
tracted on  the  shaker  with  200  cc.  portions  of  the  followirig  strengths  trichlora- 
cetic acid;  0,2  per  cent.,  1.0  per  cent.,  2.5  per  cent.,  5.O  per  cent.,  and  10.0 
per  cent.  Eight  24  hour  extractions  were  made.  Each  of  the  combined  extracts 
ware  mads  up  to  a definite  volume,  filtered,  and  total  nitrogen  determined  in 
triplicate  portions.  The  results,  expressed  as  percentage  of  the  total  nitrogen 
of  the  feed  were  as  follows; 


OZ^:bL 


:jr- 


-r  ■’ 


>t  -•.  .-c-.iH  . ■'  ai:oIi&*‘. '• 

.'  j^'T'il/V*  r::-v 

t:r  i ?"3|157  • j.  cave,’'. 

il  . 

:-r.v»-  ..  V-"  ■• 

. • I 't  Xjs? 


-ra^ 


"■  r 


- _ ^ .,  - , . •■•,  ,*.-.  ' : - --SO  *1.'  cs^  : va  “I  . iv.iv i‘ 

*"  . ' • oi ioC-” . • .^•'-1  *•  C' , 

,;■*«,  i .' ',  3 '..-■ ‘irx^  aOy^'.'*f? ''  '.£^^'.jiyiJ■^lA  j,*. 

.-f  v.ijf-J-'V;  :l1;  'l'*  V *>j  ir'*  '.a! 

- ' - - *;  ' ‘ 

w , i . ^ , _ ■ - ..  ^.  ■^.1  , J 

, i>  '.  i i •? 


l"  t.J  ’.  :' 1 -i-'C. 


r -: 


i'i'ti.C'  ra...  7 ■ ..i.",.  I 


.,  . ;:  ,xi  :'^9  ; 


'.Oii'J  VX9  V - 

■'■1 


r\ 

i'i 


a-jr-.r  :.- 


f-vi , 


-7.r 


> 


: :.  - y j ■• 

^':  - c -j;  c • ^ 

'5  *K' 

it-  ■ ; -..: , -'tcv 


:.,  I 'y: 


r Ca^  ^*Zi  -'j 

;V-']  .'•  lo  i*nof:if‘c  .-t  >C'a  < : ::o 

-3tq  . . ' •.  *^ 


_ 


H &»4W<X-'Xv  iyS** 


I f.  »»  • 1* 


, , : ’ -.:  :o  01 ; ^ 

- ’,  :.*,c  7'-:, 

)T  c'lft- 


' i • >1  i.  "nw  . i-c  : 


-14- 


Per  cent,  trichloracetic 
acid  used  for  extraction 


Per  cent,  of  the  total 
nitrogen  extracted 


O.cO 

II 

I' 


16.139 

16.053 

16.359 


1.0 

II 

II 


la .855 

lh.805 


^■5 

II 

II 


13 -WS 

13-333 

13.639 


5.0 

II 

l< 


15.355 

15.556 

15.556 


10. Q 

II 

II 


16.528 
16.639 
16 . 7c2 


A portion  of  the  1 per  cent,  trichloracetic  acid  extract  was  precip- 
itated with  colloidal  iron  and  the  percentage  of  the  total  nitrogen  of  the  feed 
left  in  the  filtrate  was  11.111  per  cent.^  11.454  per  cent.,  and  11.593  per  cent. 
Since  various  experiments  had  shov/n  ttat  the  nonprotein  nitrogen  content  of  this 
sample  of  oats  was  between  10  and  11  per  cent,  of  the  total  nitrogen  of  the  feed 
and  since  the  1 per  cent,  acid  extra.cted  all  the  nonprotein  nitrogen  and  less 
protein  nitrogen  than  ai'iy  other  strength  trichloracetic  acid  used,  the  1 per  cent, 
trichloracetic  acid  was  chosen  s,s  the  extraction  liquid  for  the  nonprotein  nitro- 
gen in  the  future  experirsents. 

(4)  Sepa  ration  of  protein  and  nonprotein  nitrogen. 

As  was  stcted  above,  the  selection  of  a suitable  precipitant  for  the 
protein  rraterial  that  is  extre.cted  with  the  nonprotein  nitrogenous  constituents 
has  been  a most  difficult  problem.  Some  of  the  points  which  must  be  taken  into 
consideration  when  choosing  the  protein  precipitant  are  as  follows:  (1)  The  re- 
agent should  contain  no  nitrogen.  (£)  The  precipitation  must  be  quantitative. 


■VV, 


i 


4- 


u 


.1  i»xn3 

jl 


' , N 


* H 


•'  i»  / 


I? 


>feV, 


^ , -.  '•.  ^ w.  •- 

fi...  Tfl"  ;C  i fr>  : ,r';-.'Xi>  ■?  i '.i'uJ.'j 

' -'3  '1C  ;.K-a  ••  - 

f*  ■ ..Cl  ’ i’ sac> 


ft  r 


■■;-3  • 


i.  , 


S','--:  •.' 


...  -..1  I ::o- 


-1 


(3)  The  precipitant  must  not  precipitate,  occlude,  or  adsorb  any  nonprotein 
nitrogenous  constituents.  (U)  The  filtration  must  be  fairly  rapid  and  practicable. 
(5)  The  reagent  must  not  interfere  in  any  way  with  the  application  of  t'he  Van 
Slyke  analysis.  Some  of  the  protein  precipitants  and  methods  of  precipitation 
tried  are:  (1)  neutralization;  (c)  neutralization  and  boiling;  (3)  10  volunes  of 
alcohol;  (4)  neutralization  follov/ed  by  precipitation  with  10  volumes  of  alcohol; 
(5)  trichloracetic  acid;  (6)  Almen's  tannic  acid;  (7)  meta-phosphoric  acid; 

(S)  zinc  sulfate;  and  (9)  colloidal  ferric  hydrate.^ 

It  would  be  useless  to  record  the  results  of  all  these  tests  but  it 
may  be  said  that  no  reagent  tested  was  entirely  satisfactory.  Hart  and  Bentley 
(10)  in  their  study  of  »' the  character  of  the  water- solr.ble  nitrogen  of  somscommon 
f eedingstuff s, » precipitated  the  proteins  which  were  soluble  in  hot  water  by 
slightly  acidifying  and  boiling  for  a few  moments.  This  method  does  not  precipi- 
tate all  the  proteins  extracted  by  the  cold  1.0  per  cent,  trichloracetic  acid, 
which  is  used  in  this  work  for  the  extraction  of  the  nonprotein  nitrogen. 

In  some  cases  precipitation  by  neutralization  followed  by  precipita- 
tion with  10  volumes  of  alcohol  serves  very  well.  By  this  method  the  solution 
is  neutralized  with  sodium  hydroxide  solution,  then  made  slightly  acid  with  ace- 
tic acid,  boiled  for  a few  minutes,  and  allovifed  to  stand  over  night,  ijihe  solutior 
is  then  filtered  and  washed.  The  filtrate  is  concentrated  under  diminished 
pressure  to  a small  volume  and  precipitated  by  the  addition  of  10  volumes  of  ab- 
solute alcohol.  This  solution  is  allov;ed  to  stand  over  night,  filtered  and 
washed  with  90  per  cent,  alcohol.  Amorig  the  objections  to  this  method  and,  in 
fact,  any  method  in  which  strong  alcohol  is  used  as  the  precipitant,  are  a slight 
precipitation  of  starch  which,  when  present  during  the  hydrolysis  of  the  protein, 
causes  an  increase  in  the  humin  nitrogen  and  the  fact  that  strong  alcohol  precip- 
itates some  nonprotein  nitrogenous  substances. 


o 


Iron  Dialyzed  Merck,  containing  5 cent.  Fe^Og. 


r-j-  ^ 


i: 


' .V  ^ 


J :«  ■ 


:-,^;jUi;;  ;■ 


U¥  '^Arf 


.or.  ..  .‘tojo*:;  ^.'r 


. :^-':  i -.i- 


' V f:  • 

: :■:••.  lUA 


4.*  -X  - 


\ ,'J  'iv  (.30, 


•»  £.*14  I . 


f»  f •r  ‘ 

4»  » « 


I 


.. 


ti  * *1 " * ; 


; .1 


j : to.  , 


!.'  O, 


; - u t . .JV  .i 


r; 


Uf  f»r*  .'.  _■  ' i'  : . .0,;-r:::iu5lY; 

,.'  : T ^ ’ ' : ' ; r.ni 

■•P”!  Oil$  •/  - .-i/-,  '■  J » .»  ; 

^ jocv.-tst  Ou  5:-.‘-;  '--i.  sr*  ’Viw- 
. . .i  i.,  ..  -I :v,c  »•..•?■'•  ii>  \l-:f  'ritu’ii  ..  : ,'vJ^ 

c ..  .■.•Ti  o-:j  ic  '. 

" .4 

% 

.;■.  .•  -.t.-  r-4  rr  1 Xt'‘ 

, J VC-- '■  ' .A  *■  fc: ; £.1?  i*  &'I‘.  '■  .'  . 1 -s^C .'"J 

. c.  - S;  . *;v . »♦.  :^v*  - . ,’  ..i  >j,:  >::■  it. 

■•  • j:4.“  ('.O'*-  ^ 0 .i  *•  ':! 

I -I.-  .r^-,  . . 'r.  i.-  9 ..  'i,  .'v- 

r'-'l  . ^ 4..:..X'''.-  ■ ' 

‘ ..  . ■ V ....  „''t  . 

-.'.  'I  '•  '■  * 

. •.  ..J  ■.' ...  r ......  x v r;  ctL.  . 

..  ;^,i‘-  . ’ -.  i 'o-v.  -.  a.;  ,;io»  »l  . . ■ •.wsiA 

.;.  , ..  . .'V‘  . . -.  iv(j 

. . j . ^''T.  • ■■\A1  --  - . ^.'  . : --''v 

*'  ■ • • •■V  . “D  i. r-  -Xv  4 


^.S.  ' 


1-.J  'C 


. : ,.  r jri 
; J i ii5  I i Otf  1 0.'^  ' * ► 
’ :;  ts-'J.Ll'r  r^’ .c  -' 


'.‘  a ; 


,.  . ) 

■A 


I i.i  :;  .t-  l»- 

, ,.x’.  c',.  .■jr.;:  t-LiO.-  ■ iJJr:  . 


L 


0.  IrtO  ? ’i.~  '-'i' 'v-Jt.SS'-:.. 


-«5T-r  J*r— r i 


..-  „ ?i 


-16- 


Gr09rayald  (IS)  has  shov/n  that  trichloracetic  acid  makes  a quantita- 
tive separation  of  the  nonprotein  and  protein  nitrogen  in  blood  but  it  has  been 
found  that  trichloracetic  acid  is  not  nearly  as  good  a precipitant  for  vegetable 
proteins^  encountered  in  this  work  at  least,  as  it  is  for  animal  proteins.  In 
addition,  the  trichloracetic  acid  precipitates  of  the  cold  1 per  cent,  trichlora- 
cetic acid  extracts  i^ave  been  found  to  be  very  difficult  to  filter.  This  is  also 
the  chief  objection  to  meta-phosphoric  acid  recommended  by  Folin  and  Denis  (19) • 
Wolff  (20)  ha.s  shown  that  colloidal  ferric  hydrate  is  a suitable  re- 
agent for  removing  the  proteins  from  blood  in  order  to  obtain  a filtrate  for  the 
quantitative  determination  of  free  amino  nitrogen.  Hill  (21)  states  triat  by  the 
addition  of  the  proper  ainount  of  colloidal  iron  all  the  proteins  of  milk  are  com- 
pletely precipitated  and  caii  be  rapidly  filtered  off  leaving  a perfectly  clear 
colorless  filtrate.  Van  Slyke,  Vinograd-Villchur,  and  Losee  (22)  make  the  fol- 
lowing statanent  in  regard  to  the  use  of  colloidal  iron;  »'In  experiments  on  Witte 

peptone  and  partially  digested  proteins  we  have  found,  furthermore,  that 

colloidal  ferric  hydrate  not  only  lets  all  the  airiino-acids  go  through  into  the 
filtrate,  but  that  it  also  precipitates  none  of  the  int errnediary  products  up  to 
the  albumosss,  and  none  of  these  except  som.e  of  complexity  but  little  below  that 

of  the  original  proteins " Grindley  and  Eckstein  (9),  in  their  study  of  the 

nonprotein  nitrogenous  constituents  of  feeds,  used  colloidal  ferric  hydra-te  to 
precipitate  the  proteins  from  their  nonprotein  extract.  From  the  results  of  many 
experiments  it  seems  that  colloidal  iron  is  not  as  good  a precipitant  for  vege- 
table proteins  as  it  is  for  animal  proteins. 

In  order  to  determine  the  proper  amount  of  colloi'dal  iron  necessary 
to  precipitate  the  proteins  from  the  nonprotein  nitrogen  extract  the  following 
experiment  was  carried  out. 


c J-: ! 


.;aur  A tier,  c : 

r ;..^/<  I -1  i'f:*  . 

. ,.,  j g. 


I*-  ;:  - A-oc : ' . i .*.<•«  . 


"i  "■  1 


'•■  p : *■; . 


si 


. ^ 0 "•joIu 
“•'i/D.'T 

I «i..7  at  ivn'if-3.’-UOO' 
t .*  * 

-V-*-.  ill ' -i,  . 


> : 


* 

r.;jt 


^ ;A  >;1< 


f- 


I 


-o  < 


'.  1. 


.So  ^ tit  y 


•'TS'i  ; :r^  .oXXo*  . .'’ 

/ 


•’*  t'T  9tS* 


' I 


i:. 


s.  ^ 


;v.  v '.  *l'i  - t-'.’ffili'  ■•• 

•r  IT  ..1  . 

: . .!  ■ ."  "3  • ® • - ' 

^ 


' » • .t.- 


1 


( 


a.  * -VI, 


I 


.i  - 0-  . 
; ^ . ••  T«*  ■ 


) 9J  f/  r 

beti^ 


■•  ■ J-'  itf-’irJi  5 

' ‘ * i{i#q  opq 

. r JiC'ztL: 

, >'>f i/j  ‘-•j/J  ^ 
. iAfxtV^s.  'ii:  ' I 


'.0  .#^>1  • ' ' ' ■ «*i i»0 , *4lOi*l  - .^i 


*1 


■■ito  i 


i 


ft  I . M iCtS 


riA  1^'i  «:  t;  . . 

V -.  *!  - a ; ' t ,.  *}  •;  a d J tiM  Ttr  - 

. 0 b.'xa-isi 


-17- 


EXPERI?/IEI'IT  V. 


Three  5^  sajuples  of  finely  ground  corn  v/ere  transferred  to  centri- 
fuge bottles  and  extracted  7 times  v/ith  dOO  cc.  portions  of  1 per  cent,  trichlora-- 
cetic  acid.  The  extracts  were  combined  and  made  up  to  6000  cc.  Five  hundred  cc. 
portions  were  used  for  each  of  the  precipitations.  The  method  of  precipitation 
in  general  was  to  bring  the  solution  to  boiling  then  to  add  the  colloidal  iron 
drop  by  drop.  During  the  boiling  and  precipitation  frequent  addition  of  hydro- 
chloric acid  were  necessary  to  maintain  the  slight  acidity  necessary  for  precipi- 
tation. After  boiling  for  1 minute  the  magnesium  sulfate  solution  v/a,s  added  and 
the  solution  again  boiled  for  1 minute.  The  solution  ws,s  then  removed  from  the 
flame  and  the  precipitate  allowed  to  settle.  The  solution  was  then  filtered  or, 
if  it  was  to  be  treated  again,  it  was  brought  to  boiling  and  the  procedure  repeat- 
ed. After  filtration  the  precipitate  wa,s  washed  thoroughlv  with  hot  water.  The 
followirig  table  gives  the  times  precipitated  before  filtration,  the  amounts  of 
colloidal  iron  used,  the  temperature,  and  the  average  results  of  nitrogen  left 
in  the  filtrate.  The  results  are  expressed  as  percentage  of  the  total  nitrogen  of 
the  saciple. 


Times 

pptd. 

cc.  of  colloidal 
iron 

cc.  of  magnesiULii 
sulfate  soln. 

Temper- 

ature 

IT  in  f ii  trate 
as  percentage 
of  total  N 

1 

25 

3 

cold 

9.67 

1 

5 

1 

boilir^ 

6.05 

a 

5.5 

1.1 

II 

7.27 

3 

5>5.5 

1.1,1 

II 

6.70 

1 

10 

1 

II 

7.69 

2 

10,10 

1.1 

6.S£ 

3 

10,10,10 

1,1,1 

II 

5.95 

1 

£0 

3 

n 

0.36 

£ 

£0,10 

3.3 

11 

5.52 

3 

£0,10,10 

3.3.3 

II 

5.I4O 

3 

10,10,10 

3. 3.3 

II 

5-i4£ 

The  filtrate  from  the  last  two  precipitations  were  reprecipitated. 


• fca  v’enil  y 


.-  ■•■ 

\K:  oi  9;  ikBff 

. I|  -<v. 

.'V  V:,  tiov-.'ro  •!<  ■ t. 

. "r-'  -v.  Oi' 5-es 

-•"  . -1* 

Sv'  r -rTyf/i  *icl  £:1  ■ ' >" 

^ Vh .'  < • i 

• •-• » i.  - • - - 

^ ■ . * ' *T  I:  » 

ic.:  ..'r  • ^ ^ ,:  ' ■'  '.  .i'l'r  ^ 

, «ii*-  lA'iSfjftjij  r.L 

\ 

. . 4 'y  •-  -.  -■! 

'-  ■ ; 

inov;  r::-i  ^ -»n 

. 'Ol  .'■-.v-t 

-1^';  ’‘*i. '*?»•? 

. ;■,,  :: 

* *i.“  ■■  -u  ’.-..T;-.'.  : 

•.• ; O'"'  ilvcl'f:, 

‘ “ ' /,>  *i  . 

. r$r.':.  . ’• 

•:.,  • - ■ . , '•■-‘’t  Silli  r-^. 

4 ' w *.  . Ty  » w 5 % 

c ■ ■;  -.r  - 

' V.  -r*  ^ 

••:.*'j^J  • i<"T' 

j i-  * -■;  • li  'i  ..j • 

' \ 

■•  f i;  .Itfie  . 

i 

, ■ fiA/-  .-.t  J.: 

.. ..  :-::c  . 

*d  ."^J  rf.lfW’-'?!  'it 

'■«^^'rc.  »r.  ,ic^:  ' 

r-.u  ' jijt ■ -I  -CJ-  i^.'*  frOV  Ifi  iSl  ' ^ ’fch^Oilvi 
, ' iixj.  ■'  ■ 'i-i 


• . !^*  r»  ^ « • .'.  i ^ r #(  • -;-?■? 


;■  -..I 

T -'  #<i  '.1 


'"'S  ' v' 


-lii- 


using  exactly  tiie  same  amoxmts  of  reagents  as  were  used  for  the  first  precipita- 
tion. The  percentages  of  total  nitrogen  reiaaining  after  the  second  precipitation 
were  4. ’/I  Pei*  cent,  and  4.?0  P®!*  cent.^  respectively. 

Tliis  experiment  indicated  that  the  best  method  of  precipitation  v/ith 
colloidal  iron  was  three  10  cc.  portions  of  colloidal  iron,  followed  by  three 
3 cc.  portions  of  magnesium  sulfate  solution,  and  allov/ing  the  precipitate  to 
settle  between  additions  of  the  colloidal  iron. 

Experiment  V,  as  well  as  others  have  indicated  that,  under  all  con- 
ditions of  precipitation  tried,  colloidal  iron  did  not  precipitate  Vne  proteins 
from  the  cold  1 per  cent,  trichloracetic  acid  extracts  quanti tatively . In  addition 
it  had  been  almost  impossible  to  obtain  results  which  a,gree,  even  on  four  samples 

of  the  same  feed,  of  the  same  weight,  run  at  the  seme  time,  and  in  as  near  the 

same  manner  as  possible.  The  reason  for  this  lack  of  agreement  was  thought  to 

be  that,  under  the  conditions  for  precipitation,  it  was  practically  impossible  to 

control  very  accurately  the  acidity  of  the  solution.  This  is  due,  of  course,  to 
the  fact  that  trichloracetic  acid  in  alkaline  solution  readily  breaks  up  on  boil- 
ing, according  to  the  reactions, 

(1)  g.Clg.COgH  + NaOH  = C.Clg.COgNa  + H^O 
(a)  C.Clg.COglTa  + H^O  = G.HCI3  + NaHCOg 

The  chloroform  vola.tilizes  and  the  reaction  goes  gradually  to  completion  towards 
the  formation  of  carborate.  According  to  the  method  of  precipitation  used  in 
the  previous  experiments,  during  the  boiling  and  precipitation  with  colloidal 
iron,  the  solution  v/as  kept  acid  to  litnms  by  the  addition  of  hydrochloric  acid 
as  fast  as  the  solution  became  alkaline.  In  8,n  attempt  to  find,  if  possible, 
more  suitable  conditions  for  the  precipitation  v;ith  colloidal  iron,  experiment 


VI  was  carried  out. 


y 


04 


-K- 


\' 


■ -vq 


j>  ' 


.'  -■  ' 


«irfP 


:or  I 


'»-*Q  < 


'a.  Ir  • ^*'  {_ 


tU 


it:'<j  . 'Ui:  **  •> 


/I*:-  •,  ••^.;jf. 


T « 


' rf 


n ' • ' 1 r - 1*  c . ' .w  - - - 

: T >C  - •■  a ''■ 


p.  ' 


0.  -:tfj 


■■  <“  i 


••■ . « jjji 


« . A 


X . 


••  4. 


. f.  j . " 

cl'  1 *:■•'  . '^1;-- 

• ..:  Iri*  . : ■*  :... 


-,A,CW  \ r 


J'  .i»  ’ ^ 1 *'■•3 


'4 

t 

Tf 


n.c'j.’  ~e^v 


IT  -n©.,_ 


: , ,.i  Ditx  t:  'f'-t'r'li'*; 


laftT 


■!*  ;.;•  T3;;'::X  i': 


4-  JSl* 


ill 


— y’ ' 


, 


I : * ‘-f 


*c..  Til: 


mJi  5 * '♦■ 


r-» 


r*.  • 

•1-  , - i i V'-  ti** : 

. ;i.-  . : : ' f’3  Jrisift  j ,;v:i  t ^oi  * “ i M -' 

. , .;i  . .'ii'-'  - --*  5 '3  i.  - 

"?’**'* 

' r y '’•<“  ■ ■ * ' •■*-'  •'^-*  ^ * t:* 


^ * -i' ! i ' 


-19- 


EXPERIivlENT  YI. 

Four  25  samples  of  corn  which  had  been  ground  to  pass  through  a 
UO  mesh  sieve  were  transferred  to  centrifuge  bottles  and  each  extracted  7 times 
v/ith  200  cc.  portions  of  1 per  cent,  trichloracetic  acid.  The  extracts  were  com- 
bined, made  up  to  bOOO  cc.,  and  filtered.  Tnis  stock  solution  was  used  for  the 
different  methods  of  precipitation  outlined  below.  Total  nitrogen  determined  in 
three  I5O  cc.  portions  gave  an  average  of  Id. 126  per  cent,  of  the  total  nitrogen 
of  the  feed  in  the  solution. 

(a)  IdUTRAX  IvIETHOD.  Tw'O  ^00  cc.  portions  of  the  stock  solution  were 
precipitated  ’with  colloidal  iron  by  neutralizing,  boiling,  and  using  three  10  cc. 
portions  of  colloidal  iron  as  described  for  experiment  V.  After  filtration  on 
Buchner  funnels  the  precipitates  were  washed  thoroughly  with  hot  water. 

(b)  DIRECT  I-ISTHOD.  Without  neutralizing,  two  5OO  cc.  portions  of 
the  stock  solution  were  precipitated  with  colloidal  iron  by  boiling  and  using 
three  10  cc.  portions  of  colloidal  iron  in  exactly  the  same  manner  as  the  above 
except  that  the  trichloracetic  acid  was  not  neutralized.  The  precipitation  was 
carried  out  in  tire  presence  of  approximately  1 per  cent,  trichloracetic  acid. 

(c)  AFTER  DECOlviPOSITION  OF  TEE  TRICHLORACETIC  ACID  BY  BOILING.  It 
had  been  noticed  that,  during  a second  precipitation  with  colloidal  iron,  the 
acidity  could  be  maintained  more  nearly  constant  tlian  during  the  first  precipita- 
tion. This  was  presumably  due  to  the  fact  that  most  of  the  trichloracetic  acid 
had  been  broken  down  by  the  boiling  during  the  first  precipitation.  Previous 
tests  had  also  indicated  that  little  or  no  hydrolysis  of  protein  is  caused  by 
boiling  with  dilute  trichloracetic  acid.  Decomposition  of  the  acid  by  boiling 
before  precipitation  was  therefore  tried.  Two  5OO  cc.  portions  of  the  stock 
solution  were  neutralized  with  sodium  hydroxide  solution.  They  were  then  boiled 
gently,  keeping  the  solution  .just  acid  with  dilute  hj^drochloric  acid,  until  there 


- -:si  ja. 


• ‘ r <T*  ^ 


ii 


a ,r-,-0‘.'7  i; -i  . 


\J0  . 


*;:t.  . 


a.rt*5  6 


, . f-  : !'J 


• . r'\'x:"\o  iof: 

e ' i-'l  jL  r 04  *:  • .'tvtB  .-u.c*u;  ‘ 

*«t  ?.-.7  r.' i ■ . ''  79q~XT6  ,s.."0.  .'ic^  .-"■'vO-  >■*'» 

, . . i • f . 't  ’ • ‘-  s’  ^ <*  ^ ^ 

\x,  ^:  ;o  c ■'.j*-.  ^;-‘'V 

r iJ  ‘j*  , , 


..X  B.:  i..: 


-■'■■  «..l*  *r>  : ■•■'■ 

. Liii*  ,jj  . /v"  , ;u  X i-z.. 


,.:r"  ' ) 


:.  :'^Ox.-:3 


•:  ' 


">■■ ,- '-  •-'  •'• 


•s  30.  «J  .V'  -.''  '. 


: * i r'4'j  h-t'f: 


V 1 r . J 


it-.  •‘r.; 


t'. 


1 


-■  itf-  .-rs-;;  - . 


f t 


■<1 


.'1^.  .-/vt  ^ 


i-.l-fc  ft-.aw  ..  i.'x.  fo« ' ‘ .'  | 

d2#1S  . t r-juv 

. : ..i  j 


: *.  ii.'  _ ‘ ' .0-t  «.i< 


. hlOi  . 

A '.-  - 


i.'  -t ;i'*a  • - ■' 

•’•*  ■ ■ . '■  . '■  1T!'''T '•  ft  t -t' 

.’*.  .Ci*  'R-r  ’ : 


;s  i.t- iTi.i- 

"'"r’/Tv- 


il,  ■ 


. ^ iV.  . • 


L • T 


crc  ^ 


■-’ i’ t .-,x-c 


* «.  gv.  4--  - 


& vf 


.5  in 


»3^iVC. 


I 


.{  . 


f , V * -U  ---U-  X.  * •-  w . .4#  . . *W  ^ •-  * f X . •- 

1 

...  :v'.  • '•..  ..T  Jj  ; . c~-  3 :.  ‘'C‘ 

3 . ■ £.,*.  f ^ ; 1 ' 6 1 n 

i :"'t i;  f-r-  • il  .'  • : 


.::/:  «.3.iii  I ’ -iJ:'  a<e  7'._ 


- i 7 -■  X :■  ' - ' ‘ ■:- ■ ‘ ‘ I ^ 


was  no  change  in  reaction.  This  took  about  IiO  minutes.  The  solutions  wore  then 
precipitated  w^ith  three  10  cc.  portions  of  colloidal  iron  as  usual. 

(d)  BY  THE  USE  OF  A BUFFER  SUBSTANCE.  It  wa-s  thought  that  by  the 
use  of  a buffer  substance  the  acidity  could  bo  accurately  controlled.  The  mono- 
basic sodium  acid  phosphate  was  selected  as  the  most  promising  from  a considera- 
tion of  its  properties  and  the  following  reactions; 

(1)  C.Clj.COgH  + NaOH  = C.Clg.CO^lfe  + E^O 

(2)  C.Clg.CO^Na  + K^0  = CHCI3  + NaHCOg  (on  boiling) 

(3)  NaHCOg  + NaK^PO^  = Na^H  PO^  + 

(4)  H2CO3  = H3O  + CO3 

An  excess  of  the  phosphate  was  considered  necessary  to  insure  a slight  acidity. 

One  5OO  cc.  portion  of  the  stock  solution  v/s,s  exactly  neutralized  to 
litmus  with  N/2  sodiiom  hydroxide,  the  exact  amount  being  noted.  Then  twice  its 
equivalent  of  mono-basic  sodium  acid  phosphate  added.  The  solution  was  then  pre- 
cipitated with  colloidal  iron,  using  tliree  10  cc.  portions  as  in  the  above  cases. 

(e)  SECOl'JD  PRECIPITATION.  Since  a second  precipitation  has  alv;ays 
removed  considerable  nitrogen  in  previous  experiments,  each  of  the  above  fil- 
trates were  reprecipitated  v/ith  colloidal  iron  using  three  10  cc.  portions  of 
colloidal  iron  as  usual. 

The  filtrates  from  the  above  precipitation  were  each  made  up  to  a 
definite  volume  and  total  nitrogen  determined  in  triplicate  aliquots.  A condensed 
table  of  the  averages  of  the  results,  expressed  as  percentage  of  the  total  nitro- 
gen of  the  feed,  follows; 


Ileutral  metuod 


Conditions  of  precipitation 

In  presence 
of  1 pet.  tri- 
chloracetic acid 


After  boiling  in  presence 


>f  NaH.PO. 

^ 4 


B 


aver. 


B 


aver. 


B aver. 


aver. 


IO.U5O  10.13^5  10.294 


After  first  precipitation 

10.410  10.585  10.49^ 


11.500  10. 780  11.140 


17.436 


6.261  5.602  5.932 


After  second  precipitation 

5.492  5.376  5.684 


5.657  6.124  5.891 


7.703 


Fto21  the  above  results  the  following  conclusions  v;ere  drawn:  (1) 
The  use  of  the  mono-basic  sodium  acid  phospliate  to  maintain  the  acidity  is  not 


practicable  for  it  interferes  in  some  way  with  the  precipitation.  ^2)  ^.'?hils  I 

boiling  the  solution  to  bree-k  down  the  trichloracetic  acid  does  not  seem  to 
cause  hydrolysis  (unless  colloidal  iron  precipitates  some  hydrolytic  products)  I 
the  method  is  less  desirable  tlian  either  of  the  first  two.  (3)  It  is  irnpossiblej 
from  the  results  alone,  to  choose  one  from  the  other  of  the  first  two  methods. 

As  to  the  technique  involved  in  the  two  methods,  the  first  or  neutral  method  is 
slightly  the  more  preferable  because  of  a quicker  settling  of  the  precipitate, 
more  rapid  filtration,  and  a clearer  filtrate.  (4)  The  second  precipitation  in 
tnis  experiment,  as  in  all  other  cases,  shows  tiiat  some  nitrogenous  substances 
are  further  removed.  This  seems  to  indicate  that  the  precipitation  of  the  pro- 
teins from  a cold  1 per  cent,  trichloracetic  acid  extract  of  the  common  feeds  is 
either  not  quantitative  or  that  it  is  removing  some  nonprotein  nitrogenous  sub- 
stances , 


The  work  of  Wolff  (20),  Hill  (21),  Yan  Slyke,  Vinograd-Villchur, 
and  Losee  (22),  and  of  others  has  proven,  however,  very  conclusively  tliat  col- 
loidal iron  does  not  precipitate  the  norprotein  nitrogenous  substances,  at  least 
from  animal  extracts.  Although  the  nonprotein  nitrogenous  compounds  found  in  an 


I J 

t 

i 


I 


f 


I :r  ^ ; 'to 


I 


: -I.’ 


I 


K 


I: 


V ~ f ^ ^ I 


?-'  'Jr  , ; - , . . : ‘ !,f  "io  0«.'  f'-T- 

. U •.  ,C«>'.UV'- 

i-' '"-J  • 4f.’.3 

- - : ;v,.c-::v  :•  «>:.v,  ■ ••  i *l  i:  '-  : . 'ti  - 

i . . -r  i'  > . " C ; i 

..-,...  v ^ ■■  '..liac  »?«9- < 

V t *•.  t-  - • • ' ■ •■‘'•''*  ' ■■ 

o . • 

* *"  ■ *“  C ^ 

■ ' . » , j 1 Tr". . 

r.  ,>,v  . 

0;  •;?  iiv  . :’  i '4 


i 0 - - ^ ■ • ■•  - ’ ’’  & ti.' , 

: --il.pf  6Mfi  f -• 


. ‘V  f ' . J v.»- 

♦ . « 

^ ,li  Jbi'.V’*:  f-' 


:*;;ji  - I* 


I 

•i  ’ . ; >T*  ■ ^ I C- ■ '•  ^-  • 

t . • - -.  . — 

I . , ■■.  ■ . 

'■  , V • r-  ‘ * I ■ ' . ' ^ 1 


■ ; r •• ; ' ^ 0,  - s <^ -- 

■ V'-  . • a . . r/3  TO  ^7 !j  :*rMDp  t'lr.  : 


..C'f 


- -.  3L..* 


. ; ^5 


/ I'Jl'J''?  V.’ 

i ,•  -.  -•-7*'.  • :o  '- 

r -. . » *.  j 0 3 >1 ' . ':i ,', eT'i'  ' 


, ' , .)  •«'  ic*«x 


issm 


aniuial  extract  should  not  differ  to  any  great  extent,  at  least  in  regards  to 
size  of  the  molecules,  from  those  found  in  a vegetable  extract,  the  following 
experiiTient  was  carried  out  in  order  to  siiow  whether  or  not  free  Stfn.r.onia  nitrogen 
or  free  amino-acid  nitrogen  was  precipitated  by  colloidal  iron. 

EXPERIIvIENT  VII. 

Two  UO  gm.  samples  of  alfalfa  and  two  oO  gm.  samples  of  corn  were 
extracted  in  centrifuge  bottles  with  ether,  alcohol,  and  1 per  cent,  trichlora- 
cetic acid  in  the  usual  manner.  The  free  ammonia  and  free  amino-acid  nitrogen 
in  the  filtered  trichloracetic  acid  extracts,  in  the  filtrates  from  the  first 
and  in  the  filtrates  from  the  second  colloidal  iron  precipitation,  were  deter- 
mined in  a.  maniier  similar  to  that  used  by  Grindley  and  Eckstein  (9).  Tne  re- 
sults, expressed  as  percentage  of  the  total  nitrogen  of  the  feeds,  v/ere  as 


follows; 


In  1 pet.  trichlor- 

After  1st 

After  ^rnd 

acetic  anid  extracts 

pp  tn. 

pptn. 

alfalfa 

1 • g64 

2.097 

2. 996 

Free  ammonia  N 

corn 

3.050 

3.034 

al fal fa 

10.749 

9.  315 

9.044 

Free  amino  IT 

corn 

2.936 

2.7S5 

2.400 

The  above  results  indicate  that  colloidal  iron  does  not  carry  down 
free  amij^onia  nitrogen  or  free  amino  nitrogen  unless  slight  hydrolysis  is  taking 
place  at  the  same  rate  of  precipitation  which  is  not  very  probable.  From  the 
fact  that  40  minutes  boiling  of  the  1 per  cent,  trichloracetic  acid  extra.cts 
without  neutralization  in  Experiment  VI  did  not  cause  any  higher  nonprotein 
values  than  was  shown  in  the  extracts  that  were  not  boiled  it  does  not  seem 
probable  that  dilute  trichloracetic  acid  will  lij'’drolyze  proteins.  In  order  to 
prove  conclusively  that  this  is  true,  Experixaent  VIII  was  carried  out. 


-^3- 

EXPSRII'.ffil?r  VIII. 

A sainple  of  oats  was  extracted  in  the  cold  a few  times  with  0,'d.  per 
cent  sodium  hydroxide  solution.  A large  amount  of  protein  was  obtained  by  neutra] 
ization.  The  precipitate  was  filtered  off  and  divided  as  accurately  as  possible 
into  3 parts.  Part  A v;as  transferred  to  a flask  with  100  cc.  of  aamionia-free 
water;  part  to  a flask  with  100  cc.  of  d.O  per  cent,  trichloracetic  acid;  and 
part  to  a flask  with  100  cc.  of  5 cent,  trichloracetic  acid.  Each  solu- 
tion was  refluxed  for  h hours.  The  free  amino-acid  nitrogen  in  each  solution 
was  determined  by  the  Van  Slyke  nitrous  acid  method.  The  results  were: 


Pressure 

Temperature 

Volume 

A,  Boiled  with  ammonia-free  water 

752.8 

di.5 

0 . 125CC . , 0.  l30c< 

B.  " •'  2 pet.  trichlorace- 

tic acid 

II 

11 

0.1c:5cc.,  0.125c< 

C.  v;ith  5 pet.  trichlorace- 

tic acid 

II 

II 

0.l30cc.,0.l30c( 

Tile  results  show  no  indication  of  hydrolysis  with  d or  ^ per  cent, 
trichloracetic  acid  by  boiling  for  'd  hours. 

(5)  Procedure  finally  adopted  for  extraction  of  noi^protein  fraction 

Tne  best  method  of  extracting  the  nonprotein  nitrogenous  constitu- 
ents of  a finely  ground  simple  of  feed,  as  shown  by  the  above  experiments,  is  7 
extractions  of  the  sample  in  a centrifuge  bottle,  first,  with  100  cc.  portions 
of  anhydrous  ether;  second,  200  cc.  portions  of  cold  absolute  alcohol;  and  third, 
200  cc.  portions  of  cold  1 per  cent,  trichloracetic  acid.  The  small  amount  of 
protein  material  extracted  by  the  trichloracetic  acid  is  remioved  by  precipitat- 
ing with  colloidal  iron.  For  this  precipitation  the  extract  is  brought  to  boil- 
ing, nade  distinctly  aLkaline  with  sodiun  hydroxide  solution,  then  immediately 


-^4- 

made  just  acid  to  litmus  with  hydrochloric  acid.  Tsn  cc.  of  colloidal  ferric 
hydrate  are  slo'wly  added^  the  solution  boiled  for  1 minute,  3 cc.  of  a solution 
of  magnesium  sulfate  (made  by  dissolving  crystallized  magnesium  sulfate  in  an 
equal  volume  of  water)  are  added,  and  the  solution  boiled  for  1 minute.  The 
solution  is  then  removed  from  the  flcime  and  the  frecipi  tS'.te  allov.'ed  to  settle. 
During  the  boiling,  precipitation,  and  standing  the  solv.tion  is  kept  acid  to 
litmus  by  the  occassional  addition  of  hydrochloric  acid.  As  soon  as  the  precip- 
itate has  settled  the  solution  is  again  brought  to  boiling  and  the  precipitation 
with  10  cc.  of  colloidal  iron  and  3 cc.  of  magnesium  sulfate  solution  repeated. 
After  the  precipitate  has  settled  the  second  time  the  procedure  is  again  repeat- 
ed. After  the  precipitate  has  settled  the  third  time  the  solution  is  filtered 
on  iiard  paper  v/ith  suction.  The  precipitate  is  v;ashed  thorou^ly  with  boiling 
aranonia-free  water.  The  filtrate  is  now  reprecipitated,  using  three  10  cc.  por- 
tions of  colloidal  ferric  hydrate,  in  exactly  the  same  manner  as  described  for 
the  first  precipi tetion. 

B.  TREATiOTT  OF  THE  RESIDUE  INSOLUBLE  IN  ETHER,  ABSOLUTE  ALCOHOL, 

AND  COLD  1 PER  CEN’T.  TRICHLOPJLCETIC  ACID. 

In  the  improved  method  of  Eckstein  and  Grindley  (I5)  the  residue 
left  after  extractions  v/ith  ether  and  alcohol  was  digested  for  I5  hours  three 
or  four  times  with  0.1  per  cent,  hydrochloric  acid  until  all  the  starch  liad  been 
converted  into  sugars.  The  residue  insoluble  in  0.1  per  cent,  hydrochloric  acid 
was  boiled  with  20  per  cent,  hydrochloric  acid  until  the  proteins  v;ere  ccanpletely 
hydrolyzed.  The  proteins  in  the  0.1  per  cent,  hydrochloric  acid  extract  were 
precipitated  by  neutralization  and  by  5 volumes  of  absolute  alcohol.  The  pro- 
teins precipitated  by  neutralization  and  by  alcohol  were  completely  hydrolyzed 
by  boiling  with  20  per  cent,  hydrochloric  acid.  The  filtrate  from^  the  alcoholic 
precipitate  was  finally  hydrolyzed  by  boiling  S hours  with  5 cent,  hydro- 


1 


-25- 

chloric  acid. 

The  first  crianga  to  be  made  in  the  Eckstein  and  Grindley  method  was 
the  complete  removal  of  all  the  nonprotein  nitrogenous  constituents.  The  proce- 
dure finally  adopted,  as  outlined  in  the  last  section  above,  consisted  of  extrac- 
tions in  the  centrifuge  bottle  with  ether,  absol^ite  alcohol,  and  cold  1 per  cent, 
trichloracetic  acid.  The  small  amount  of  protein  material  extracted  by  the  tri- 
chloracetic acid  was  recovered  by  precipitation  with  colloidal  ferric  hydrate. 

The  next  was  a minor  change  and  consisted  of  \ising  10  volunies  in- 
stead of  5 vol-umes  of  absolute  alcohol  to  precipita.te  the  proteins  remaining  in 
the  0,1  per  cent,  hydrochloric  acid  extract  after  precipitation  by  neutralization. 
Ten  volumes  of  absolute  alcohol  was  found  to  remove  the  proteins  more  completely 
and  at  the  same  time  less  starch  tlian  was  precipitated  vd.th  5 voliimes  of  alcohol. 

With  these  modifications  it  was  possible  to  separate  the  nonprotein 
nitrogen  and  the  greater  part  of  the  carbohydrates  from  the  main  portion  of  the 
proteins  before  the  latter  were  hydrolyzed.  In  this  manner  the  quantity  of 
htimin  formed  was  reduced  decidedly  in  the  case  of  cereals,  but  only  slightly  in 
the  case  of  rougiiages.  This  latter  fact  has  also  been  noticed  by  Eeidig  and  Sny- 
der (17)*  Tlie  explanation  of  the  fact  that  the  httmin  nitrogen  of  roughages  is 
decreased  but  little  over  that  obtaixied  by  the  direct  hydrolysis  of  the  feed, 
used  earlier  by  Grindley  and  associates,  is  probably  because  the  roughages  exaa;in- 
ed  had  comparatively  little  starch  and  a high  content  of  fiber  v/hich  is  present 
during  the  hydrolysis  by  the  Eckstein  and  Grindley  method.  The  fiber  present 
during  the  hydrolysis  is  the  proba.ble  cause  of  the  high  humin  nitrogen  values. 

In  later  experiinents  this  has  been  shown  to  be  the  case  or  at  least  experiments 
in  wnich  the  fiber  was  not  present  during  hydrolysis  show  much  lower  nitrogen 
values. 


jl 


■%■■:  ' 


.•  ' .•.'  I *r  ■ ■-  JO  cl  ^.  ■; 


. ' I iocn  e 


■ r; . . ■'  J . ‘ ^ 


■i'  • *>■?•. 


i.  A ■ 


•I  -T: 


' : J.,1  ••;  :.co2'io  Jj 

» T d iw  C*  MM.;  * ’ 


I 


•e  ..  i-c«  V ^0  l).3eJ 


i--. 


t.--/  i . 


I 


1.  '^i  i.  '^■‘  -5 

-. . ~ 1 c.. ": . ■ zi  li. 


. § . 


a.;T.-.j->  aol 


:..J  ■.•?-  r-rvj^i,**' 


H 


♦ ^Ufr 


'' , ^ ^ c 


i - 


J . -.-'X  ..  -.'  '•44:;^  ' ., 

45;^*; • . <j' 


A*  itm.-  ' -u  .'• 


' ■">  •*•::  ~ <* 


AC  .?  x;-  ./ 


M **r 


...  ■ ■•:  -■  .:  . :>  ■ 

.1.-  • 1 - 'IJ  ■ ' * 

V"ti  r::!.  .. 

. :r-i‘’'='  ' ■ '^■*  9-- 

' ?r  - . f-I'. 

..  Jx-  J-oc ’^oe-- 

'.  . . ••  i - * M . ;^; . ' ^v  .<.  •'.  -te  ‘ 

‘co-iojo  . >evn JrA-r-ia^-  i 

» -.Z-"  •‘  O - ■•  -C  . - - ‘~ 


-j  c 


i-..-  . V-  rr.  : u-: 


1"  »'  ..  . 


.V'.-.  ..  3;.'-  j5.il'  3r.3jl 


CV  i J 3.1  t 


Z j,SZ,>  ,tj. 

. '/iJ6% 


-^0- 


EXPERIMEIfT  IX. 

In  order  to  remove  the  starch,  fiber,  and  other  substances  which 
might  interfere  with  the  application  of  the  Van  Slyke  analysis,  many  changes  were 
tried.  The  first  was  a dilute  alkali  extraction  of  the  residue,  insoluble  in 
ether,  alcohol,  and  1 per  cent,  trichloracetic  acid,  to  remove  most  of  the  pro- 
teins. The  strength  alkali  used  was  O.h  per  cent,  sodium  hydroxide  solution.  Sia 
HOO  cc.  extractions  in  a centrifuge  bottle  were  made,  two  extractions  being  made 
each  24  hours  as  usual.  At  first  attempts  were  i.oade  to  precipitate  the  proteins 
extracted  by  the  dilute  alkali.  Neutralization,  trichloracetic  acid,  and  col- 
loidal iron  were  all  used  for  this  purpose  but  a small  amount  of  nitrogen  e.lways 
remained  unprecipitated.  Farther  tests  showed  that  no  substances  which  inter- 
fered with  the  Van  Slyke  analysis  were  extracted  by  the  dilute  alkali.  The  alka- 
li extract  was  therefore  made  distinctly  acid  with  hydrochloric  acid,  concentrated 
under  diminished  pressure  to  a small  voluine,  transferred  to  a digestion  flask 
with  an  equal  volume  of  concentrated  hydrochloric  acid,  and  completely  hydrolyzed 
by  boiling  under  a reflux  condenser  for  I5  to  20  hours. 

The  residue  insoluble  in  dilute  alkali  was  then  digested  vrith  0.1  per 
cent,  hydrocnloric  acid  and  the  sterch  removed  in  the  usual  raa.nner.  Two  ^0  gm. 
samples  of  oats  which  had  been  ground  so  as  to  pass  throu^  an  80  mesh  sieve  ’.\^re 
extracted  completely  with  ether,  absolute  alcohol,  cold  1 par  cent,  trichilorace- 
tic  acid,  cold  0.2  per  cent,  sodium  hydroxide,  and  0.1  per  cent,  hy'drcchloric  acic  , 
Tne  residues  reiiiaining  after  the  la.st  treatment  were  transferred  to  Kjeldahl 
flasks  and  the  total  nitrogen  determined.  Four  and  fourteen  hundredths  per  cent, 
and  4.3^  cent,  of  the  total  nitrogen  of  the  feed  remained. 

Two  more  3O  gm.  portions  of  the  same  sample  of  oats  were  treated  in 
the  same  manner  as  the  two  above  with  the  exception  that  after  the  extraction  of 
the  starch  with  hot  0.1  per  cent,  hydrochloric  acid  the  residues  were  transferred 
back  to  the  centrifuge  bottles  and  extracted  with  200  cc.  portions  of  strorig 


, fc.  : . ,noxJ.' : iU  o.ij  AJ  ’ <5'*CF'iTe -V‘;i  i •. 

rr:  o:  v.  r-  ^.■ -->'iCfro  LlAiiiit  c,  - - r.  ; i " 

t-  ' . . ; O^’  iv' '.  : : •*  ' . \ *i«.,  I **;  ' r . 


Af  ?*r:f.  li/--  - jvi  c*  . ■.:  ..:  -*1  '-I^'  net!'- . X' ; 

1.  ..  . -.  . »r  •- ' <.J  //.v:  'tSW':'..  I.:  7 


u .r'A 


^ i:  .' 


‘:c . 


^DX*lJX-‘5  ii 


r :■_  •’...  '.Ir-  •■  i , -O'-’  iv.  jfc  r:.A  zl'i  '* 

. :..  .::  . 

i.  , -.  . ■ *c  - 1 

I . ■.'•'■  . ©t'j  ni  1’- 

ij -■ ';i  -'.  ilL  ;.ij;  ;;./-  li  f y f ; 

I 

- c iJvr  ;•■./  . i - Icn  ; 

. ■■’•  *'  •••.•-•  . . . . n ■ . 

d . ^ 

. • t Z ?.  w L * ' *.C  f-  /’ 

I ■ ■ ■ 

, 7;]  j,<- j .-.  TT  .*  • ^'..  . . c '-■■-■ 

:( 

• 1 

'■  ; ;•>] . o r•r'^x::  : 3i  1^jqo\ 

J 

■ toT--'..  la:#  U^x  tzs  0-' 

WJ''-:*4r  : i • " . "■■  . * L'./ 


DO' /c.  . tt?  rc  i^'V  ax 

. :■  .i:»7sfrcL  x_:.  irv  . ' 

,/j  4> >,  ' „.-5  ...  ■ ^'>.  * r "’*'’i5a  t Di  t —a  * 

0.'  ;--it  <•  .'  O-  />:  .'D  ■ 

..  i-LVX~  .It  'v  - i i“iV  J I ■’  .?  4.0  .i  : 

A,  4-j‘,LL:aAA  ^-T.  ♦&  r.’:  ■'..  -'li  a 

'..  yvl-.'X  ..  .•■*  Dt  , -oi  iro  j.  'Ice  .'  lO*  .c/i. 

v.yj 

’/O':*  J-.  - t.  - . :.-;ii  «:i  er** 

I 

. ' . .J--. /r  r.-''*"’  • ‘ 

*:o  ■:  • <5  - . b'.* 

t In  . 0,,  <■■  . cr?  ^ 

‘ ?'a;  'j,i'  Dvoc';  o*»^J  o.  -*  AcriiX.'...-  c -Aa  *dl  , 

''i,.'  . . , . *lv  . ■'  .1  I ? i ■ .iiiOSM  J 

.AA  r=  i7‘>  } ;’^i DJ «j  .'  o- 


-d1- 

alkali.  Each  extraction  was  for  a cil4  ho'or  period.  The  first  four  extractions 
v/ere  w'ith  O.d  per  cent,  sodiui:  hydroxide^  the  fiftii  v/ith  O.5  per  cent.,  the  sixth 
with  1 per  cent.^  and  a seventh  with  5 P'^r  cent.  The  two  residues  insoluble  in 
strong  alkali  were  transferred  to  Kjsldahl  flasks  and  total  nitrogen  determined. 
l.Sll  per  cent,  and  1.30c;  per  cent,  of  the  total  nitrogen  of  the  feed  remained. 

Three  more  3O  gm.  portions  of  the  SO  mesh  oats  were  extracted  vrith 
ether,  alcohol,  cold  1 per  cent,  trichloracetic  acid,  cold  O.c;  per  cent,  sodium 
hydroxide,  and  hot  0.1  per  cent,  hydrochloric  acid  in  the  usual  manner.  The  resi- 
dues were  then  transferred  to  round  bottom  digestion  flasks  and  boiled  3 minutes 
with  C5O  cc.  portions  of  dO  per  cent,  hydrochloric  acid.  The  solutions  v/ere 
cooled,  filtered,  aiid  washed.  The  treatment  with  dO  per  cent,  hj/drochloric  acid 
was  repes-ted  again.  After  washing  the  residues  thoroughly  they  v;ere  submitted 
to  Kjeldahl  analysis.  Tv;o  and  one  hundredth  per  cent.,  d.d'k  per  cent.,  and  ^.37 
per  cent,  of  the  total  nitrogen  remained.  Tae  dO  per  cent,  hydrochloric  acid 
extracts  were  made  up  to  a definite  volume  and  total  nitrogen  determined  in  ali- 
quot portions  showed  tiiat  c.40  per  cant.,  d.yd  per  cent.,  and  <i.51  per  cent,  of 
the  total  nitrogen  had  been  extra,cted. 

Since  tv;o  3 •minute  boilings  v/ith  'dO  per  cent,  hydrochloric  acid 
removes  about  2.5  pei'  cent,  of  the  total  nitrogen  and  a strong  alkali  extraction 
about  3 per  cent,  of  the  total  rhtrogen  remaining  in  the  residue  insoluble  in 
ether,  alcohol,  cold  1 per  cent,  trichloracetic  acid,  cold  0.2  per  cent,  sodium 
hydroxide,  and  hot  0.1  per  cent,  hydrochloric  acid,  both  extractions  v/ere  made  on 
two  50  gra.  samples  of  oats  and  tv;o  3O  gm.  samples  of  corn.  The  c;0  per  cent, 
hydrochloric  a.cid  extraction  was  follov/ed  by  the  strong  alkali  extraction.  The 
percentages  of  total  nitrogen  in  the  residues  v/are  0,120  per  cent,  and  O.IO3 
per  cent,  in  the  case  of  oats  and  O.I32  per  cent,  and  O.II7  per  cent,  in  the  case 
of  corn. 


From  the  results  of  the  above  experiment  the  20  per  cent,  hydrochlor- 


1 


1 


-28- 


ic  acid  digestion  follov/ed  by  the  strong  alkali  extraction  was  adopted  as  part 
of  the  regular  procedure.  Tne  20  per  cent,  hydrochloric  acid  extract  was  trans- 
ferred to  a digestion  flask  with  a volume  of  concentrated  hydrochloric  acid  equal 
to  the  volume  of  the  water  used  in  the  washing  which  had  been  kept  separate  from 
the  20  per  cent,  hydrochloric  acid  extracts  and  measured.  This  gives  a 20  per 
cent,  hydrochloric  acid  solution  and  the  proteins  in  the  acid  extract  are  com- 
pletely hydrolyzed  by  boiling  I5  to  20  hours. 

At  first,  precipitation  of  the  proteins  extracted  by  the  strong  alka- 
li was  attempted  but  finally  this  extract  v/as  acidified  with  hydrochloric  acid, 
concentrated  under  diminished  pressure,  and  hydrolyzed  with  20  per  cent,  hydro- 
chloric acid  just  the  same  as  in-the  case  of  the  dilute  alkali  extract.  Later 
the  extraction  v;ith  three  5O  cc.  portions  of  5 per  cent,  sodium  hydroxide  for 
24  hour  periods  'was  adopted  as  tiie  procedure  for  the  strong  alkali  extraction. 

It  was  found  that  this  extraction  removed  just  as  much  nitrogen  as  the  more 
elaborate  method  of  four  0.2  per  cent.,  one  O.R  per  cent.,  one  1 per  cent.,  and 
one  5 cent,  sodium  hydroxide  extractions.  This  smaller  volume  of  extract 
was  much  more  desirable  than  the  larger  ’volume  because  of  the  smaller  quantity 
of  salts  introduced  into  the  final  hydrolysate  ard  also  because  the  strorig  alka- 
li extract  is  very  difficult  to  concentrate  on  account  of  the  foaming.  The 
smaller  volume  of  extract  can  be  neutralized  and  without  concentration,  if  nec- 
essary, tra.nsf erred  to  a digestion  flask  with  an  equal  volume  of  concentrated 
hydrochloric  acid  and  hydrolyzed  in  the  usual  manner. 

Qualitative  tests  with  3O  gm.  samples  of  oats,  corn,  and  alfalfa  in- 
dicated that  by  the  use  of  the  above  procedure  the  quantity  of  humin  was  not 
large  and  that  during  the  precipitation  of  the  bases  witli  phospho tungstic  acid 
only  a very  small  aaiOunt  of  black  substance  was  formed  alorig  with  the  bases. 

C.  EXTMCTION  OF  STARCH  WITH  TRICHLORACETIC  ACID. 

All  the  extractions  in  the  procedure,  as  modified  according  to  the 


. r i.* t . X • ' v) " ■•'  ■'  - ^ ’ i ^ J ^ ^ i — — 

_ ■ ^ *•-  .viiJ  itv::;  , 

- - *-rft  i'  €i  'i'  ‘4^;::  •.’■i 

'r  i-  . ri  . -'  •; *'».:  •**•  . •‘- r :■'•..  .. 

: ■ ^.  J ;J‘-  • .■-■••iOiq  » 

i 

A 

f.  ; " 

. . ' . . V..V  . . " ■ 'J  -:j.  . i?  f v ~ I 

; ;i  s.'-ijl'v  T:IiA. . . cT : . ~ui<^  . •:.‘w;  r.-Z''  ^ 

i 

.:-.*•  I'i:  ■ ‘1C  - . '■'^c  -..t  .....  J -.i  ..  ».:c'.?!  C--  _ Js 

- r- 

'■  J oii^LUf^  '■  : . . ' -f  ■ "ji  i-  ' ..  '-V  -'v  i t;;S..’_f  cc 

. t . ; *!o  ..  -..I  f t/.c^  * '-..'Oi  :'\X'  *’  ■.  cl  - vt  ■2i3;c,.  i)j.  j 


*» 


-•i?:.-  ".1’  ,.  1‘  3 ..tl 


n 


u 


i 


\{ 


I 


, .Oi..; r ■.  j.  I c.  '■•a  ,v'  i'A*'  . * ' i'  . 

1;;' c.  c ‘ic-  ' i- « o:  . c'j;  ‘ , c- . 

. c.  ..!  ■' ' c\' ' *’■  •' iC  ; 

. > -s:  - : . '''"  -r  . " ■ .■•  • ^ '■■•-:  ' ...^- 

0 ■ ■ n....  oi  . ? .. 

_ j, 

.:a.  , .>•.  .[.jkJ  . J iu  . ; AT-ic  v...  • ,..;c-.‘'.  JAli 

I- 

’ *r 

; . • ■ «r  J • lUiiftXrf  ■ -11  - : V > ^ 


r* 


> 


7 "'Vi': 


'T-  V 


\ 


. ♦ 
4Ak 


if 


t 


fx 


-rN 


• j 

H 

\ 

U 


r. 

i 

t 


'.  -JCS 


last  section,  were  now  quite  satisfactory  v/ith  the  exception  of  the  extraction 
of  starch  with  hot  0.1  per  cent,  hydrochloric  acid.  Tliis  extraction,  as  carried 
out  by  the  Eckstein  and  Grindley  method,  consisted  of  boiling  the  residue  in- 
soluble in  ether  and  alcohol  for  I5  hour  periods  with  0.1  per  cent,  hydrochloric 
acid  until  the  residue  gave  no  starch  test.  Two  or  three  I5  hour  digestions  were 
usually  necessary  to  remove  the  starch  completely  from  the  cereals  such  as  oats 
and  corn.  The  proteins  extracted  by  this  procedure  were  precipitated  first,  by 
neutralization,  and  then,  after  concentration  of  the  filtrate,  with  5 volumes  of 
absolute  alcohol.  Two  to  5 cent,  of  nitrogen  usually  remained  unprecipitated. 
The  filtrate  from  the  alcoholic  precipitation  was  concentrated  under  diminished 
pressure  and  completely  hydrolyzed  with  5 cent,  hydrochloric  acid  by  boiling 
6 hours. 

As  was  mentioned  in  a previous  section,  5 volumes  of  alcohol  does  not 
precipitate  proteins  as  completely  as  10  volumes.  This  change  w'as  made  but  still 
there  reirained  considerable  nitrogen  in  the  filtrate.  Colloidal  iron  was  used  but 
with  no  better  results.  Vfnen  tne  alcdiolic  filtrate  ws,s  concexitrated  and  finally 
hydrolyzed  even  with  5 pe^’  cent,  hydrochloric  acid  and  boiling  for  only  <3  hours 
quite  a large  aiiount  of  humin  resulted. 

Since  the  proteins  could  not  be  precipitated  completely  free:  the  ex- 
tract containir:g  the  hydrolyzed  starch  and,  of  course,  there  ms  the  possibility 
of  slight  hydrolysis  of  protein,  it  was  thaught  that,  if  it  were  possible  to  ob- 
tain a reagent  which  would  dissolve  the  starch  without  hydrolyzing  it,  it  might 
be  possible  to  precipitate  the  starch  from  the  proteins.  It  is  'well  knes-vn  that 
certain  organic  acids  will  dissolve  starch  v/ith  little  if  any  hydrolysis.  For 
example,  a 1 per  cent,  salicylic  acid  solution  will  dissolve  starch  and  the  solu- 
tion filters  quite  readily.  Dr.  Mitchell  suggested  the  use  of  a ci  per  cent, 
trichloracetic  acid  solution  in  an  autoclave  and  the  precipitation  of  the  starch 


-30- 


with.  two  volumes  of  95  P^r  cent,  alcohol.  Since  it  W5s  fomd  that  trichloracetic 
acid  dissolved  only  a small  amount  of  protein  in  the  extraction  of  the  nonprotein 
nitrogen  the  follov/ing  test  expsriiiient  wa.s  carried  out  to  test  the  solvent  ac- 
tion of  trichloracetic  acid  on  starch. 

EXPERIMENT  X. 

Two  10  gm.  samples  of  oats  v/hich  had  been  ground  to  pass  tnrough  an 
SO  mesh  sieve  were  completely  extracted  with  ether^  absolute  alcohol,  and  water 
and  O.OS  per  cent,  hydrochloric  acid  in  centrifuge  bottles  in  the  usual  raamer . 
The  residues  were  labeled  A and  B.  Residue  B v«.s  then  extracted  6 times  v/ith 
<£00  cc.  portions  of  alkali  in  the  centrifuge  bottle.  Each  residue  was  then  trans 
f erred  to  a I5OO  cc.  round  bottom  digestion  flask  v/ith  '^0  cc.  of  h per  cent, 
trichloracetic  acid.  The  first  idea  was  to  heat  in  the  autoclave  but  it  was 
thought  later  tliat  the  saiue  thing  might  be  accomplished  by  heating  on  the  steam 
bath.  About  two  thirds  of  solution  A va.s  transferred  to  a separate  flask  and 
labeled  A1  while  the  remaining  portion;  was  labeled  A2.  Solution  A1  was  heated 
in  the  autoclave  for  I5  minutes  at  I5  pounds  pressure  while  ML  was  placed  on  a 
steam  bath,  and  with  frequent  sliaking,  allowed  to  digest  until  it  was  apparent 
(from  the  disappearance  of  the  milky  color)  that  much  of  the  starch  had  been 
dissolved.  This  took  about  45  minutes.  Solution  B was  set  aside  until  the 
tests  on  solutions  A1  and  Ah  had  been  made.  Solutions  A1  and  A2  were  filtered 
on  hard  mper  with  suction  while  hot.  Tbi.e  filtration,  while  quite  rapid  in  both 
cases,  was  faster  in  solution  Ah.  The  residues  were  washed  a,  few  times  with  hot 
water  and  then  tested  with  potassium  iodide  solution  for  the  presence  of  starch. 
Residue  A1  gave  a slight  but  distinct  starch  test  while  residue  Ak  gave  no  test 
for  starch.  The  filtrates  v/hile  hot  were  v/ater  clea,r  but  on  cooling  became  milkj 
and  after  ste-nding  over  night  much  starch  had  settled  to  the  bottom  of  the  flasks 
In  order  to  insure  complete  extraction  of  all  the  starch  the  two  residues  were 


-31- 

transfsrred  back  to  tneir  original  digestion  flasks  with  I5O  cc.  of  d.  per  cent, 
trichloracetic  acid.  A1  was  again  placed  in  the  autoclave  for  I5  minutes  at 
15  pounds  pressure  and  Ac  heated  on  the  steain  bath  for  about  3O  minutes.  At  no 
time  during  the  second  digestion  of  .A2  did  it  give  a starch  test,  while  A1  after 
being  removed  from  the  autoclave,  gave  a very  distinct  test.  The  solutions  were 
filtered  and  washed  as  before.  The  filtrates  were  added  to  the  corresponding 
filtrate  from  the  first  filtration.  Solution  B was  then  treated  on  the  stemn 
bath  in  exactly  the  same  manner  as  Aci.  The  3 filtrates  v;sre  warmed  until  the 
starch  dissolved  and  then  filtered  to  remove  any  solid  material  that  might  have 
got  into  thei'a.  After  cooling,  each  filtrate  was  made  up  to  1000  cc.  and  total 
nitrogen  determined  in  duplicate  aliquots.  The  results,  expressed  as  percentage 
of  the  feed,  were; 


Al  - treated 

in 

the 

autoclave,  not  extracted  with 

di  lute 

NaOH 

16-5, 

14.8  pet. 

A2  - treated 

on 

the 

steam  bath,  » »'  '• 

II 

II 

3.8, 

4.0  " 

B - treated 

II 

11 

" " , extracted  ” 

II 

It 

2.0, 

1.7  «' 

T’.vo  cOO  cc.  aliquots  of  the  filtered  extracts  were  treated  with  'd 


volumes  of  95  cent,  alcohol  to  precipitate  the  starch.  After  standing  3 days 
the  starch  precipitates  were  filtered  off  on  ijard  paper  with  suction  end  v/ashed 
with  alcohol  {^d  volumes  05  cent,  alcohol;  1 volume  of  v/ater"^.  Each  of  the 
sterch  precipitates  ware  transferred  to  Kjel^d^il  flasks  and  total  nitrogen  de- 
termined. The  duplica.tes  were  averaged  and  showed  0.10  per  cent.,  0.12  per  cent, 
and  0.10  per  cent,  of  the  total  nitrogen  of  the  feed  in  the  starch  precipitates 
Al,  A2,  and  B,  respective!;/. 

The  res'olts  from  the  above  experiment  indicate  that  very  little  ni- 
trogen is  extracted  by  hot  2 per  cent,  trichloracetic  acid,  especially  if  the 
greater  part  of  the  protein  material  has  been  removed  previously  by  a dilute 
alkali  extraction;  that  the  extraction  of  starch  can  be  carried  out  very  quickly 


-32- 


and  easily  by  heating  on  the  steam  bath  for  a comparatively  short  period  of  time; 
and  that  two  volumes  of  alcohol  precipitates  most  of  the  sts-rch  without  precipitat  > 
ing  but  little  nitrogen. 

In  order  to  compare  the  total  nitrogen  extracted  by  hot  0.1  per  cent. 
h3'’drochloric  acid  and  hot  c;  per  cent,  trichloracetic  acid  the  following  experi- 
ment was  carried  out. 

EXPERLMEITT  XI. 

Four  16  gm.  samples  of  30  mesh  oats  were  transferred  to  centrifuge 
bottles  and  extracted  in  the  follo'wing  manner:  Two  samples,  A and  B,  were  ex- 

tracted with  two  250  cc.  portions  of  ammonia -free  water,  followed  by  two  cc. 
portions  of  0,02  per  cent,  hydrochloric  acid,  which  in  turn  were  followed  by  2 
more  25O  cc.  portions  of  amraonia--free  water.  Two  extractions  were  nade  each  24 
hours.  Two  samples,  C and  D,  were  extracted  6 times  v/ith  200  cc.  portions  of 
1 per  cent,  trichloracetic  acid.  Two  extractions  were  mads  each  24  hours.  (See 
Experiment  III  for  this  part  of  the  experiment.  Experiment  III  sdiowed  that  the 
water  and  0.02  per  cent,  hydrochloric  acid  extracted  27.975  cent,  of  the  to- 
tal nitrogen  of  the  feed  while  the  1 per  cent,  trichloracetic  extracted  only 
14.575  cent.).  The  residues  were  then  extracted  6 times  vvith  cold  dilute 
sodium  hydroxide  to  remove  most  of  the  proteins.  Residues  A and  C were  then  di- 
gested with  2 per  cent,  trichloracetic  acid  on  the  steam  bath  until  the  starch 
was  removed  in  the  manner  described  in  the  preceeding  sxj)eriraant.  Residues  B 
and  D were  extracted  in  exactly  the  same  rnamier  as  A and  C with  the  exception 
that  0.1  per  cent,  hydrochloric  acid  was  used  instead  of  2 per  cent,  trichlorace- 
tic acid.  The  extracts  were  made  up  to  a definite  volume  and  total  nitrogen  de- 
termined in  duplicate  aliquots.  The  avererage  results,  expressed  as  percentage 
of  the  total  nitrogen  of  the  feed,  were; 


j 7 - f V i 


: P-r:  .li,  ‘ -'f-f 

"I’lq  X i.:c'5  C « te  i . ■ ■ . . . 


V,. 


11.  :^tf 


.-i  '7>  . L-.  ’ivr.  -•-  .rOT, 


: r.  I ' . : o J '->  . ' ■:  : -.  v ? c l “f 

" ^r, 


fVlOA  fHo'l.lt.'T;! 

. r'xvifiS  LXJ'  ?.- 


■ -'I 


art 


i f “-'i  * ■ 


l«"OW  /■- 


X>. 


on*.,  -.t.  . . u::-  c;.  , • i.  - ' 

-1  J 1 ■ '!  n-TJAt  r ':«..•»  ,ii  trn 

. ,r-:<.  a.i  < If.  , lliioi . c..^i7; -••  < . . c;; 


i OVM  Iw  I'ff  c - r-r : 
. J'  (iilOl-'l'j 

- .•'N;;  - ■ •' 


■.J  X ; J- 


':.  7\7.;  1 1.^?  TTl 


.~.KL.  •'i.,' . •-  . „ . '-  ' - '■ 

t I . *-  •'  .v4.>  *c«  : b£ 

. . . • ■ . :7  . c-fi 

. A X.  • - • • - . ...  j— *>  — ' - 1 fc/.  ‘ aO  A . 


Lo.  iXt  -on;  ;'  • 1:K 


• -»  S.  » . • 


• X . V 


'.  OJ>  '-  ^ ti ./ 


.-<2  i-'.-.'Iuu*. 


c»4!w  “•  •wVCftrfl 

..  . 7 . ''  * '.X  A *«  ; • '•■•:-■-•#?  -■':  -li  . aAtJa  e-nsw'Cl 

■cz  n-  \ '.0  ^a^c&ir!;  . < •-  - ...  tioA  o.noUcc'xf  _ . '■ 

fT' V t rr  sji.nt’ifi,  r.  ft'iitoi  tno  . k'Ccuani.  ^ fri^  ^2^  tt: 

,/crV  fc  ;.*  lo  tn  U c)  *■ 


0X4?  I ^ n i AS  2 . .-nci  X!  .■•  tfiitJIRc . 

■ ■ :;/»r 


-33- 


Total  nitrogan  extracted  v/ith  the  starch 
I ' 


I by  hot  c;  pet. 
trichloracetic 
acid 


from  the  residues  left  after  extractions 

with  cold  and  0.0^  pet.  HCl 
and  cold  dilute  NaOH 


(A)  pet. 


by  hot  0.1  pet . 
HCl 


(B)  c..<£70  pet. 


with  cold  1 pet.  trichloracetic  acid 
and  cold  dilute  KaOK 


I (C)  1.91b  pet.  I (D)  3.5Q5  pet 


Experiment  III  showed  that  over  I3  per  cent,  more  nitrogen  was  re- 
moved from  A and  B than  fran  C and  D,  yet  the  hot  h per  cent,  trichloracetic  acid 
removed  less  rutrogen  from  C tnan  from  A,  which  had  over  I3  per  cent,  more  ni- 
trogen in  it,  and  the  hot  d per  cent,  trichloracetic  removed  only  1.91b  psr  cent, 
nitrogen  from  C while  hot  0.1  per  cent,  hydrochloric  acid  removed  3.595  cent, 
from  D,  which  had  the  same  amount  of  nitrogen. 

In  order  to  determine  whether  or  no  t a second  addition  of  two  volnmes 
of  alcohol  to  the  hot  c per  cent,  trichloracetic  acid  extracts  causes  an;t’'  addi- 
tional precipitation  of  starch  and  also  in  order  to  determine  whether  or  net  the 
small  amouiit  of  protein  extracted  by  the  hot  2 per  cent,  trichloracetic  acid  was 
completely  hydrolyzed  by  boiling  the  concentrated  alcoholic  filtrate  from  the 
starch  precipitate  with  5 Psr  sent,  hydrochloric  acid  for  6 hours,  the  following 
experiment  was  carried  out. 

EXPERIivIElTT  XII. 

Five  25  gm.  portions  of  corn  w'ere  weighed  out  and  labeled  1,  2,  3^ 
and  5*  Each  portion  was  transferred  quantitativeljr  to  a 5OO  cc.  centrifuge  bot- 
tle and  extracted  with  cold  1 p?r  cent,  trichloracetic  acid,  v/ith  dilute  sodium 
hydroxide  and  with  2 per  cent,  trichloracetic  in  the  usual  manner,  iphe  trichlora 
cetic  acid  and  dilute  alkali  extracts  v/ere  discarded  v/hile  the  extracts  contain- 


■f'  ,f<  . ..  ’■  • . ■JC'-i 


; f i 


V 


*■  * •* 


b 


cJ  ■:■ 


w .1 


I&:tl 


i 


t 


i 

* 

I 


li 


ii. 


I 

I 


i 


■i  -rf  t. 


>~ 


r ti 


■ cit;.  ' jr.OTiaf  II VA 

'•  f V '• '■  0.1 


• ; » j?  ic-,.l5c 
«AJ  ;. ::/t  . • dv  w 


r *^*- 


ixCjianr#  r“"'  ;*:  ' 

4 V I 

► i'c  .oI  -'>1''- 

••il.'  _3l> 


*1 




-3U- 

ing  the  starch  were  treated  as  follows:  Each  of  the  2 per  cent,  trichloracetic 

acid  extracts,  without  neutralization,  were  concentrated  -under  diminished  pres- 
sure to  about  175  cc.  The  concentrated  solutions  were  then  transferred  to  liter 
beakers,  upon  which  marks  had  been  placed  to  indicate  cc.,  iiade  up  to  the  25O 
cc.  mark,  heated  on  the  steam  bath  until  much  of  the  starch  had  dissolved,  and 
then  500  cc.  of  95  psr  cent,  alcohol  added.  The  solutions  were  allowed  to  stand 
3 days,  the  starch  precipitates  filtered  on  Buchner  funnels  fitted  with  hard 
paper,  and  the  precipitates  washed  w'ith  a 1:2  water-alcohol  solution.  The  alco- 
holic filtrates  were  concentrated  under  diminished  pressure  to  about  ^0  cc.  One 
hundred  cc.  portions  of  ammonia-free  waiter  were  added  and  the  solutions  concen- 
trated again  to  about  ^0  cc.  This  procedure  v/as  repeated  three  or  four  times  in 
order  to  remove  all  the  alcohol.  The  very  concentrated  solutions  appeared  turbid 
but  the  turbidity  disappeared  on  dilution  v/ith  water.  Sokations  2,  3,  and  5 were 
transferred  to  dOO  cc.  beakers,  upon  v;hich  marks  had  been  placed  to  indicate 
130  cc.,  the  solutions  made  up  to  ISO  cc.,  and  3OO  cc.  portions  of  95  psr  cent, 
alcohol  were  added.  Upon  the  addition  of  about  50  cc.  of  alcohol  the  solutions 
became  clear,  just  as  was  the  case  on  dilution  of  the  concentrated  solutions  with 
water.  At  no  time  and  in  neither  of  the  samples  was  there  the  least  turbidity  or 
precipitation  of  starch.  Therefore  no  attempt  was  imde  to  precipitate  solutions 
1 and  4 a secorjd  time.  The  alcoholic  solutions  of  2,  3,  and  5 were  repeatedly 
concentrated  to  small  volumes  under  diminished  pressure  to  remove  all  the  alco- 
hol. Each  of  the  concentrated  solutions  were  then  transferred  to  5*^0  cc.  round 
bottom  digestion  flasks.  Solutions  1 and  2 were  made  up  to  I25  cc.,  the  acidity 
being  5 per  cent,  hjrdrochloric  acid.  Solutions  3,  4,  and  5 were  :is.de  up  to  200 
cc.,  the  acidity  being  20  per  cent,  hydrochloric  acid. 

All  the  solutions  were  then  placed  on  the  hot  plats  under  reflux 
condensers  and  boiled  for  6 hours.  The  current  v;as  then  turned  off,  samples  1 
and  3 removed  and  cooled  to  room  temperature  (2S  degrees  C.}.  By  means  of  an 


i. r,'.-:  ^ i • • Li:. 


!»•;  - 


«#iU 


*• tr.  Z'Z  c No  -.'  .".'..tv.  ‘ T'  V' ' 
^ .:!  (.i  i*i.:  - • ;.  .. . - . 


)1  o)  I '• 


'■i'xor  i -- ■ 

. -.-.ci  •; 


IL  -.- 


'<  • 


T ’*^  V i , -j 


p?\rJ 


I/i-8  ' 1: 

% w 


■i*.  ,■  3 " * 

■:-:I  rt  . - . . - 

. _:F:  . 


“■  -w  * S •**  ,'  U. 


.V 


-»• 


■■1^. 


-r  l^  ^-. 


•:9.  aojj;^5Xi^  s-Io  . 


U • W A 9* 


^6r  c.  f'-J 

ev  oust  i - 

^ i i-  1 - ifc  - ^ • • » 

^4)  *«/V  tiW 

C:  _ r>-7  <? 'X':  7' u -'.1 

^ N:-'  • ■■  "":l 

, .iV a “7wV( 


«su^ 


j . i‘*  . ..  o-i  er»»-.St 


. 14-j: 


■■  ’''7l?‘-_-t.‘TX;'’.'  w5i-'V  7:rf>  e'TC-'^'  --."  *■  j 

r it..-*?-  c ■ l^-' - fi/.  : : j .7^.*:?  .' . A •-'..‘■j.’ir- 

';i^■' j .:«:.  V?  7 ■•  ;3rf  : , . " - ^ ^ 7- 

~.  ri  r-lv-  ..  Ivv  «V'  i'.  ^ t .:  1.  J ..  z^Lrif  CoiSU^Xov  IX.  ^ oi  XfiJc  .J  i.z. 


..zi  : i 


_w„  >.C-.  ■.,  . 


o'lSff  ..  }—  i .«..:<7i;, 


n /tox; 


^ w*^  V ^.V  • ' 


■OMtrXoJ  , £ 1 '.  . I,,;.  o“.j  ■ 


: if ; . *^?.r  v e/ 


t ^• 


ivlf.-'!- 

«.c. 

,:o 


,,  •<  ■ 

U'  • 


— > « -d. 


• £U 


'■-w  ,j.A 

I-:':  N'.:i ■•>>:■  tt: 


z zti  z y:;* 
»* 

’ K .' 


v-'rvr ■ ' 


• * s.  *•; 


»o»»? 


accurately  calibrated  burette  two  4 cc . samples  were  ranoved  from  each  of  solu- 
tions 1 and  3.  By  means  of  the  same  burette  o cc.  of  v/ater  were  added  to  each 

4 cc.  portion.  Amino  nitrogen  was  determined  in  a 10  cc.  portion  of  each  of  the 
1<£  cc.  solutire.  In  running  the  determination  the  niachine  was  allowed  to  stand 

5 mimtes  and  then  shook  for  1 minute.  The  results  were*. 


l-I 

II 

3-1 

II 


7 41  • 5 • 

ti 


cip 


0.40  cc. 
0.36 

0.82 

0.60 


Solutions  and  flasks  1 and  3 were  weighed  and  then  replaced  on  the 
hot  plate  and  boiled  for  2 hours  longer.  The  solutions  were  cooled  to  28^  and 
any  loss  in  weight  made  up  with  water.  Amino  nitrogen  run  in  the  same  manner  as 
the  above  showed  the  following  results: 


l-I 

II 

3-1 

II 


741 . 5 nim« 

II 

11 

II 


29' 

II 

II 

II 


0.40  cc. 

0.40 

0.62 

0.62 


Tests  for  completeness  of  hj/drolysis  after  10  hours  boiling  showed 
the  following  results 


l-I 

II 

3-1 

II 


737  • ^ 

II 

II 

II 


22'- 

23' 

II 

II 


0.37  cc 
0.37 

0.56 

0.70 


Tests  for  completeness  of  loydrolysis  after  I5  hours  boiling  showed 
the  following  results; 


l-I 

737.4  mm. 

20'^ 

0.42 

II 

II 

210 

0.44 

(— 1 
1 

II 

20° 

0.59 

II 

II 

II 

0.59 

ninety  cc.  of  concentrated  hydrochloric  acid  were  then  added  to  1, 
making  a 20  per  cent,  hydrochloric  acid  solution,  and  the  solution  boiled  for  10 
hours.  Tests  for  complete  hydrolysis  showed: 


i 


lI 


y 


s 

j 


l-I 

II 


-36- 


0.70  cc. 
0.06 


7UU.U  iim. 

II  II 

The  volmna  of  triis  solution  was  IbO  cc.  while  the  volume  of  3 was 
dOO  cc.  If  the  above  results  are  multiplied  by  d00/l60  the  cc.  of  nitrogen  is 
about  the  same  as  the  amount  of  nitrogen  evolved  in  3 after  I5  hours  boiling. 

Two  facts  are  brought  out  by  this  experiment;  First,  the  proteins 
which  are  extracted  by  hot  d per  cent,  trichloracetic  acid  from  the  residues  pre- 
viously extracted  v;ith  dilute  alkali,  are  completely  hydrolyzed  by  boiling  6 nours 
with  dO  per  cent,  hydrochloric  acid.  See'ond,  I5  hours  boiling  with  3 cent, 
hydrochloric  acid  show  no  more  hydrolysis  than  b haurs  boiling.  Considering  0.60 
cc.  IJ  as  indicating  co.mplete  hydrolysis  in  a solution  of  200  cc.  then  O.I4O  cc., 
the  N evolved  after  6,  8,  10  and  13  hours  boiling  with  5 per  cent,  hydrochloric 
acio.,  in  a solution  of  1^5  cc.  v^ould  indicate  that  a 5 pci*  cent,  hydrochloric 
acid  solution  hydrolyzes  the  proteins  present  to  about  40  per  cent.  The  fact, 
that,  if  tne  5 P®-  cent,  hydrochloric  acid  solution  is  made  up  to  20  per  cent, 
hydrochloric  amid  and  hydrolyzed  for  10  hours  more,  the  amino  nitrogen  then  be- 
comes the  ssa:s  as  that  v/hich  indicated  complete  hydrolysis  in  the  other  20  per 
cent,  hydrochloric  acid  solutions,  also  indicates  that  5 cent,  hydrochloric 

acid  does  not  cause  complete  hydrolysis. 

The  constancy  of  the  amino  nitrogen  values  obtained  on  testing  the 
5 per  cent,  hydrochloric  acid  solution  for  completeness  of  liylrolysis  after  6,  o, 
10,  and  15  hours  boiling  is  the  probable  reason  vfny  Eckstein  and  Brindley  made 
the  mistake  of  considering  the  proteins  in  their  similar  fraction  completely  hy- 
drolyzed by  boiling  for  0 hours  with  5 per  cent,  hydrochloric  acid. 

From  the  appearance  of  the  solution  boiled  for  6 nours  and  the  one 
boiled  for  I5  or  ^3  hours  with  20  per  cent,  hydrochloric  acid  it  is  possible  that 
less  humin,  and  if  so  probably  less  hum.in  nitrogen,  is  fom^ed  at  the  end  of  6 
hours.  Since  hydrolysis  appears  to  be  complete  at  the  end  of  o hours  there  is, 
however,  no  use  of  boiling  for  a longer  time  in  tlie  case  of  corn  at  least.  Since 
there  is  probably  little  more  than  1 per  cent,  of  the  total  nitrogen  of  the  sample 


-37- 

in  this  fraction  for  any  feed  examined^  no  appreciable  error  would  be  introduced 
by  not  boiling  longer  than  b hours, 

Idot  only  do  tlie  results  of  Experiments  X,  XI,  and  XII  favor  the  ex- 
traction of  the  staren  with  trichloracetic  acid  over  0.1  per  cent,  hydrochloric 
acid,  but  the  ruanipulation  in  case  of  the  former  reagent  is  by  far  the  more  pre- 
ferable. Qualitative  Van  Slyke  analyses  of  oats  and  corn,  using  the  trichloraceti 
acid  method  of  removing  the  starch,  gave  basic  phosplio tungstates,  which  were  wliite 
or  cream  colored  granular  precipitates,  entirely  free  from  a,ny  black  colored  sub- 
stances. This  alone  would  v/arrant  the  adoption  of  the  trichloracetic  acid  method. 

(1)  Fir.al  procedure  for  the  extraction  of  starch. 

The  procedure  finally  adopted  for  the  extraction  of  starch  was  as 
follows:  The  residue  insoluble  in  ether,  absolute  alcohol,  cold  1 per  cent, 

trichloracetic  acid,  and  cold  0.t£  per  cent,  sodium  hydroxide  is  transferred  to  a 
1500  cc.  ro'and  bottom  digestion  flask  with  5OO  cc.  of  ^.0  per  cent,  trichloracetic 
acid  and  placed  on  a.  steam  bath.  Y/lth  frequent  shaking  the  solution  is  allowed  to 
digest  until  it  is  apparent  (from  the  disappeara^nce  of  the  milky  color)  that 
much  of  the  starch  has  been  dissolved.  This  usually  requires  about  1 hour.  The 
solution  is  filtered  while  hot  through  a Sucliner  funnel  fitted  with  hard  paper. 

Tlie  residue  is  washed  a few  times  with  boiling  v/ater  and  the  residue  again  treat- 
ed with  cc.  of  c!  per  cent,  trichloracetic  acid  for  about  a half  hour  period. 
The  hot  solution  is  filtered  and  washed  as  before  and  if  the  starch  is  not  all 
removed  this  treatment  is  repeated.  The  extract  is  concentrated  tuider  diminished 
pressure  to  about  kOO  cc.  and  then  the  starch  precipitated  by  the  addition  of  two 
voluiaes  of  95  cent,  alcohol.  The  solution  is  allowed  to  stand  until  the  pre- 
cipitate settles  (about  d days)  and  then  filtered.  The  precipitate  is  washed 
w'ith  alcohol  (h  volumes  of  95  cent,  alcohol;  1 volume  of  water).  The  alco- 
holic filtrate  is  concentrated  repeatedly  ur.der  diminished  pressure  to  remove 
all  the  alcohol  and  is  then  completely  hydrolyzed  by  boiling  8 hours  with  20  per 


cent,  hydrochloric  acid. 


-3d- 

IV.  EX'IEACTION  OF  STARCH  AI^D  NOIJPROTEIH  NITROGENOUS  CONSTITUENTS  WITH  HOT 
2 PER  CENT.  TRICHLORACETIC  ACID  AIT)  THE  SEPARATION  OF  THE  STARCH,  PRO- 
TEIN AND  NONPROTEIN  NITROGEN. 

It  has  been  shown  during  the  course  of  this  work  that  cold  1 per  cent, 
trichloracetic  acid  extracts  all  the  nonprotein  nitrogenous  compouinds;  that  the 
starch  can  be  readily  extracted  with  hot  2 p er  cent,  trichloracetic  acid;  that 
2.0  per  cent,  trichloracetic  acid  will  not  hydrolyze  protein  on  boiling  for  the 
length  of  time  required  to  extract  the  starch;  and  that  hot  2 par  cent,  trichlor- 
acetic acid  extracts  but  little  protein.  It  v/as  thought  tha.t  it  might  be  possible 
to  extract  the  sample  of  feed  directly  with  hot  2 per  cent,  trichloracetic  acid, 
separate  the  starch,  protein  nitrogen,  and  norprotein  nitrogen  and  in  this  manner 
save  much  time  and  reagents  by  doing  away  v/ith  the  ether,  absolute  alcohol,  cold 
1 per  cent,  trichloracetic  acid,  and  cold  0.2  per  cent,  sodium  hydroxide  extrac- 
tions. In  cases,  where  the  distribution  of  the  nitrogen  in  the  nonprotein  frac- 
tion v;as  riot  desired,  it  was  thought  that  the  proteins  might  be  precipitated 
leaving  the  starch  and  nonprotein  nitrogen.  The  total  nitrogen  determiriation  on 
this  fraction  would  give  the  percentage  of  norprotein  nitrogen  while  the  protein 
precipitate  v/ould  be  saved  for  ai'ialysis.  V/ith  these  id^-s  in  mind  several  exper- 
iments and  tests  were  mads  with  the  following  general  results; 

First,  if  the  residue  left,  after  extraction  with  hot  2 per  cent, 
trichloracetic  acid,  is  dried  and  then  extracted  with  ether  and  alcohol  in  the 
usual  manner,  almost  the  same  aarount  of  nitrogen  is  extracted  as  is  extracted  from 
the  fresh  sample  by  ether  and  alcohol.  Of  course,  this  amount  of  nitrogen  is  very 
small  but  the  extraction  of  fats  with  ether  h-'s  been  shovm  to  be  very  helpful  in 
the  application  of  the  Van  Slyke  ar.alysis. 

Second,  while  the  precipitation  of  the  starch  'With  alcohol  was  almost 
a.s  complete  as  in  other  cases,  as  shown  by  v/eights  of  the  starch  precipita.te,  the 


i 


-39- 


precipitate  contains  considerably  more  nitrogen  than  is  the  case  when  the  nonpro- 
tein and  much  of  the  protein  nitrogen  is  removed  by  previous  extrs-ctions. 

Tnird,  colloidal  iron  precipitation  of  the  proteins  extracted  by  a 
direct  2 per  cent,  trichloracetic  acid  extraction  leaves  an  average  of  about  lb 
per  cent,  of  the  total  riitrogen  in  case  of  cats  and  about  12  per  cent,  in  case 
of  corn.  Fran  a large  number  of  experiments  the  nonprotein  nitrogen  of  oats  and 
corn  has  been  shov/n  to  be  about  I3  psi*  cent,  and  10  per  cent.^  respectively. 

Fourth,  a colloidal  iron  precipitate  of  the  proteins  extracted  by 
hot  trichloracetic  acid  contains  very  little  if  any  starch.  This  was  shovm  by 
the  following  experiment. 

EXPERBIEhT?  XIII. 

The  starch  was  removed  from  about  20  gm.  of  finely  ground  corn  with 
2 per  cent,  trichloracetic  acid  by  the  direct  extra-ction  of  the  sample. 

A.  The  proteins  were  precipitated  fron  the  2 per  cent,  trichloracetic 
acid  extract  with  colloidal  iron  in  the  usual  marmier.  The  precipitate  gave  no 
starch  test  with  potassium  iodide  solution.  It  was  thought  that  the  color  of  the 
colloidal  iron  precipitate  might  mask  the  color  test  but  on  mixing  a very  small 
amount  of  starch  paste  with  a portion  of  the  precipitate  the  test  was  very  clea-r. 

B.  About  2 gn;.  of  the  colloidal  iron  precipitate,  obtained  in  (A) 
were  boiled  for  about  3O  minutes  with  dilute  hydrochloric  acid,  the  solution  neu- 
tralized, filtered,  and  the  filtrate  tested  for  reducing  sugars  with  freshly  pre- 
pared Fehling's  solution.  IJo  reducing  sugars  v/ere  present. 

C.  Two  gm.  of  starch  ware  dissolved  in  225  cc.  of  2 per  cent,  tri- 
chloracetic acid  by  boiling.  This  is  the  approximate  proportion  of  starch  to  acid 
in  the  usual  experimient.  This  solution  was  precipitated  with  colloidal  iron  in 

the  usual  manner,  the  precipitate  filtered  and  washed.  On  testing  v/ith  potassium 
iodide  solution,  the  precipitate  gave  absolutely  no  test  v\hile  a,  very  small  por- 


i ^ 

r‘ 

j 


'"t  . u'i 


• .if  ■ .<  » i ..J  _ . 

, ..  '.J-V;'  . 

' f'.Tf. r^:  V-  •" 


’ r’.:; i > \. 


■ rf  a 


.!  :f  ; 


0 " ; 

. 3v>isj  a.  . :o  i:  ^*.  : > 

■ . ' •■  ( 
-rr'S  -I'  ’ 


/. 


1 


»f . , * i > c ^ ^ . . _ ' ' " i 1 - ^ •.  i ^ ‘ “ ‘‘ 

:.e  i-  . - : v :r  r;  i : ,:  , i'  •:.  ; . ’•  - ^ •:  • . ^ 

^■'.  . . iv,  C_  . 'tire:  /j;  ..  . »><-  •:  . .w  ,,  . ..  'iv'  f *’’.  r 

•:.  i*,  v i'.:>  T , ::  rTT  I .i  .'IIi: ::  , * " ;c* 

. ' . -.'If  . . :.•  *: 

.tfirjXi  n»ic^  a 3w 


1**:^::'  : •.  ..  - •:  ' - ■'.  / it  0"  ■ •^ 


' 'iw.  ^ 

\ 

Mft  J : 1 


w£  .*■  c ^ r. . '"r*'  ' ' •.,  _ **  j ; • ,,v'Iv?  ‘■.".i-JiJ  J.'.iJl 


.Jkuy.r.  anA  I.  c->.  i. -.  .. 

. -!  1:  V I r::  ^ . y.j  c-  • 


Bt3  ':o^^  , 

I 


I 


i 


- . *;  iu  . . '.i  ',;;;;  • .' . > .'i.  iJi  i''  « ' ' :,'..i.rc  0^ 


( 

^L;  ; • ■’tiCiTC*  ir - 

, 


■1  , 

: £»eiiuT  ‘»*tt  /. 
.^¥•Ti_-X'3 
-. ! , i it"  - 


• ' ]^ujL/c 


V-Jlil  r . ~Ji 


ac.'  ;,.  Ijfjitp  !>: : 


, , . •,  .'ta 


J 


4 


' 1 t 


-no- 


tion of  the  filtrate  highly  diluted  gave  a very  clear  test  for  starch.  Both  the 
filtrate  and  the  colloidal  iron  precipitate  were  boiled  for  3O  minutes  with  dilute 
hydrochloric  acid^  neutralized  and  filtered.  Two  cc.  of  the  filtrate  from  the 
original  filtrate  gave  by  boiling  'd.  minutes  with  freshly  prepared  Fehling's 
solutions,  quits  a large  precipitate  of  cuprous  oxide.  The  filtrate  from  the 
hydrolysis  of  the  colloidal  iron  precipitate  was  made  up  to  ^0  cc.  and  a 5 cc. 
portion  gave  absolutely  no  cuprous  oxide  and  a 15  cc.  portion  gave  just  a trace 
when  tested  in  the  usual  rxanner  with  freshly  prepared  Fehiing's  solutions. 

?/here  tiiue  is  more  imxportant  than  accuracy^  the  direct  extraction  of 
the  feed  with  hot  2 per  cent,  trichloracetic  acid,  followed  by  the  precipitation 
of  the  proteins  vnth  colloidal  iron,  nay  perhaps  be  used  to  an  advantage.  The 
filtrate  from  the  colloidal  iron  may  be.  concentra,ted  and  the  starch  precipitated 
with  two  volumes  of  alcohol,  leaving  the  rionprotein  nitrogen,  the  distribution  of 
which  nay  be  detennined  if  desired. 

V.  MODIFICATIONS  OF  THE  VAN  SLYKE  METHOD 

In  the  application  of  the  Van  Slyke  method  to  the  analysis  of  feeds, 
the  original  method  (6)  modified  as  to  the  method  of  decomposing  the  basic  phos- 
photungstates  (23)  was  used.  Some  slight  modifications,  most  of  which  were  made 
necessary  by  the  larger  amount  of  protein  material  present,  have  been  made.  A 
few'  other  minor  changes  in  the  technique  have  been  found  advisable.  Plimmer's 
(2h)  method  and  a schenae  for  aeration  long  used  in  this  laboratory  is  used  for 
the  arginine  determination,  Denis'  modification  of  Benedict's  method  for  organic 
sulfur  is  used  for  cystine.  If  considered  necessary’-,  an  e2pla.rjation  for  any 
further  change  will  be  given  in  the  detailed  procedure  oi\tlined  below. 


-Ill- 


VI.  THE  METHOD  IN  DETAIL. 

PART  I.  PREPARATION  OF  THE  SA/IPLE. 

Twenty  five  to  100  pconds  of  an  average  sample  of  the  feed  is  obtained 
usually  on  the  rijarket.  The  sample,  except  in  the  case  of  roughages,  is  mixed 
thoroughly  and  quartered  down  until  about  a 2 to  4 gallon  sample  rerriains.  In 
case  of  roughages  the  entire  sample  is  cut  in  small  pieces  by  passing  it  through 
a motor  driven  cutter.  This  satrrple  is  then  mixed  and  qua.rtered  down  until  a.  H 
to  4 gallon  sarnple  remains.  The  2 to  4 gallon  sample  in  all  cases  is  then  coarse- 
ly ground,  thoroughly  mixed,  and  quartered  dovv-n  again  until  2 to  6 quarts  remain. 
This  final  sample  is  then  ground  so  tnat  the  entire  sample  passes  through  a 40 
to  60  mesh  sieve.  It  is  then  mixed  thoroughly  and  stored  in  air-tight  Sui’e  Seal 
jars.  The  total  nitrogen  determined  very  carefully  in  each  sample  of  the  feed 
is  the  basis  of  all  calculations  since  all  nitrogen  determira.tions  are  expressed 
in  percentage  of  the  total  nitrogen  of  the  feed. 

Four  portions  of  the  finely  ground  sample  of  feed  are  us'oally  run  at 
the  same  time.  The  samples  are  of  exactly  the  same  weight  and  the  weight  is 
always  such  that  each  portion  contains  approximately  6 gn.  of  protein.  Duplicate 
or  triplicate  total  nitrogen  determinations  are  always  made  whenever  possible. 

Each  portion  is  treated  as  fellows: 

PART  II.  PRSPAPuS.TION  OF  THE  HYDROLYZED  PROTEIN  SOLUTION. 

A.  EXTPA.CTION  WITH  AI3IYDR0US  ETHER. 

The  sample  of  feed  is  quantitatively  transferred  to  a 5OO  cc.  centri- 
fuge bottle  and  <^00  cc.  of  anhydrous  ether  added.  The  bottle  is  corked  securely 
and  the  cork  wired  in  place.  The  bottle  is  then  placed  on  a mechanical  siiaker 
v/hich  keeps  the  bottle  and  contents  in  centinusou  motion.  Two  extractions  are 
made  each  dM-  hours;  one  extraction  for  a 7 to  6 hour  period  is  made  during  the 


r 


■ V 

'V 


■r  ir  Ai/o 


- 


^ :..if  ..>*.  t-*.  V.  ,-.  &- 

c ;c  ZI  ■ t ' .._  .. 


u ^ 


r I 4 


a7 


. :t:.  ; ^ 

1 . y -I.,;.  .4  I'  'IC-0  *>i.< 

«:4‘  V ■ • ' -v  ‘wjjf  1. 

>••:;,■:  . .1*  s l»,  CU.:- i Ji.'.  1.  T'  C? ' : 


tr.j- 


•*.  - -ft  -.-V  '.'.  ?*  •'  Ja  i 

...  \ ,;i  ; .’.  I . virc  ■-'caa.  07 

•'?■.  *:••,' «i  u«jO'..  M -:cj  o.r 


» -j 


•,u : A - i.  ■ 


?r-  i Hr* '•  J 


Cx,-.  . :.■' 


• T » • -.-sn  -ft 


•-=  ^ . 


i,  -oj  lo  c /'wor'ivo  .'.*• 

I 'A  ’ V 

•-J’.  ■.■■  J -■  &>.u: -t: -.Au  j T . ' •"iij  . Dco  '%'^syf 

■ 4 • ••  ; '.I  ..I'-.-sj*’!  ■- . iti 


a-  !'t;A  ■'1;,-  -i  ne.  v" 


...  ■A*'. *■ 

A . U--  - . I,-'  i ■ '.*V^  J.*.--. 


rnA*!  ^ 


( 


• ?i  '*-1 


■ .:  ..c -.. .■  vi  ^ -.T  I . v 
• ' i.  - ' . Af'.iie.  V , 

. • I '-'-lI.  •:•■.  ,.  . .-  U -OC  ■ • . . 


U-.  ; '.<.x  rx^yr:‘-vi>.  n..: 


^ S.JT'V/  5' 


"rJ*  -■■r-o  U\i  I 


....j  y.w  • ;i  -l:;t'.  o '.;f  \ ■; 


.jxif  ^r.  : laod  icrr 


‘«ir* 


-Uii- 


day  and  a second  extraction  for  a 14  to  I5  hour  period  is  made  during  the  night. 
After  each  extraction  the  sides  of  the  bottle  are  washed  down  with  a fev/  cc.  of 
ether  and  the  solution  centrifuged  until  the  solid  material  forms  a compact  mass. 
The  perfectly  clear  supernatant  liquid  is  decanted  into  a properly  labeled  flask. 
This  treatment  with  ether  is  rex^eated  6 times,  using  100  cc.  portions  of  anhydrous 
ether  for  each  extraction.  The  combined  ether  extracts  are  filtered  by  gravity 
turoiigh  Whatimn  filter  paper  No.  1,  using  a 4 inch  funnel.  The  flask  and  filter 
paper  are  washed  thoroughly  v/ith  ether.  Any  solid  matter  collected  on  the  filter 
paper  is  transferred  back  to  the  insoluble  residue  which  is  treated  as  directed 
below  in  (B).  The  ether  filtrate  is  acidified  vath  a few  drops  of  sulfuric  acid, 
the  ether  recovered  by  distillation  from  a Kjeldahl  flask,  and  the  total  nitrogen 
determined  in  the  residue  ranaining  in  the  flask. 

B.  EXTRACTION  1.YITH  COLD  ABSOLUTE  ALCOHOL. 

Tv;o  }vundrad  cc.  of  absolute  alcohol  are  added  to  the  residue  insoluble 
in  ether.  The  bottle  is  corked  securely  and  placed  on  the  shaker.  The  time  of 
the  extraction  during  the  day  should  be  7 to  S hours  and  the  time  of  extraction 
during  the  night  should  be  14  to  I5  hours.  After  each  extraction  the  sides  of 
the  bottle  are  washed  down  with  a few  cc.  of  alcohol  and  the  solution  centrifuged 
until  the  solid  matter  forms  a compact  mass.  The  perfectly  clear  supernatant 
liquid  is  decanted  into  a labeled  flask.  This  treatment  with  alcohol  is  repeated 
5 times  using  each  time  £00  cc.  of  absolute  alcohol.  The  residue  is  washed  twice, 
using  each  tiir.e  100  cc.  of  absolute  alcohol,  placing  on  the  shaker  for  1 hour, 
centrifuging,  and  decanting.  The  alcohol  extract  is  filtered  by  gravity  through 
a vrnataan  filter  paper  No.  1,  using  a 4 inch  funnel  and  the  flask  and  funnel 
washed  with  absolute  alcohol.  The  alcoholic  filtrate  is  acidified  with  a few 
drops  of  sulfuric  acid,  the  alcohol  recovered  by  distillation  fran  a Kjeldahl 
flask,  and  the  total  nitrogen  determined  in  the  residue  remaining  in  the  flask. 


1 -V  ^ 


m 


-1 

i 


5 ' 


-c  V.  jtcifAttr-.#*’.  ■•  '- 
■'J  ■z-:^‘iarr^r*-n^  ■: 

- f 

» • * -•>«.}' 
..ifi-'  r’  .j  ..  .ijr:  . .-v  i»  ':■: 

. *.  . rl  \ t » ,:*  wC':J  ’ 


, 1 

'\ 


I ■-  *’■■'•,  , .1  f>  1*' ' 

1 


i.  ■ •-■  L**._  . ‘c:  . ;fjr.: 

.-  . »■,  ; ..  .y.-  *■  :v  -iJ., 

, tfd  :.l.  - ; * ..;  ’iS 

. .’•■.  ■ _ -".‘I  . Pi..’  1 

. .,  . ..  .-.i  tt  : Zf'^- 

t ^ r.  ■ . - Cl.-  : »* 

ac;  .•  . OOJ  „■ 


. . . vs  - 

i.0 , i - - ’ - - ■ • - * 

f'j- S' "■'/•■*;  .1  ■ '...  i—.  • I*"* 


I 


i 


i'l  5, 


h f ^ r -^  #• 


■J  S (■‘J  V c. 


. -»r 


4 


; i 


t>  ..,_ 


• * '■•  Y(- 


. w-^*» 


-\-.f 


-U3- 

Any  solid  uiatter  collected  on  the  filter  paper  is  added  to  the  insolable  residue 
which  is  treated  as  described  below  in  (C). 

C.  EXTRACTION  WITH  1.0  PER.  CENT.  TRICHLORACETIC  ACID. 

The  residue  insoluble  in  alcohol  is  extracted  in  the  centrifuge  bottl« 
with  HOO  cc . of  1.0  trichloracetic  acid.  The  time  of  the  extraction  during  the 
day  should  be  | to  H hours  and  the  time  of  extraction  during  the  night  should  be 
14  to  15  hours.  After  each  extraction  the  sides  of  the  bottle  are  washed  dovvn 
with  a few  cc . of  aujiaonia-free  water  and  the  solution  centrifuged  until  the  solid 
matter  forms  a compact  mass.  The  supernatant  liquid  is  decanted^  allowing,  if 
possible,  no  undissolved  material  into  the  decantates.  The  residue  is  then  ex- 
tracted 6 times  with  200  cc.  portions  of  1.0  per  cent,  trichloracetic  acid,  mak- 
ing two  extractions  each  24  hours.  Tne  residue  is  then  vmshed  two  or  three  timep; 
using  100  cc.  portions  of  ammonia-free  water,  placing  on  the  shaker  for  1 hour, 
centrifuging,  and  decanting.  The  residue  is  treated  s.s  directed  below  in  (D) 
and  the  extr-act  as  directed  below  in  (H). 

D.  ETI'RACTiaT  WITH  DILL^E  SODIUivI  HYDROXIDE. 

The  residue  insoluble  in  1.0  per  cent,  trichloracetic  acid  is  now'  ex- 
tracted in  the  centrifuge  bottle  w'ith  200  cc.  portions  of  dilute  alkali.  The 
time  of  extraction  during  the  day  is  7 to  hours,  using  0.2  per  cent,  sodium 
hydroxide,  and  the  tLme  of  extraction  during  the  night  is  14  to  1 5 hours,  using 
0.1  per  cent,  sodium  hydroxide.  After  each  extraction  the  sides  of  the  bottle, 
are  washed  dovmi  with  a few  cc.  of  aiomonia-free  vrater,  the  solution  centrifuged 
and  decanted  as  described  for  the  previous  extracts.  If  the  alkali  extracts  tend 
to  foam  a few  drops  of  alcohol  are  added  before  the  solution  is  centrifuged.  Six 
extractions  with  dilute  alkali  are  made  and  then  the  residue  is  washed  with  100 
cc.  portions  of  ammonia-free  water  in  the  manner  described  above  until  practically 


fraa  of  alkali.  As  soon  as  tiia  extracts  are  decant9d_,  they  are  made  distinctly 
acid  with  hydrochloric  acid.  The  residua  insoluble  in  dilute  sodium  hydroxide  is 
treated  as  directed  below  in  (E) . The  dilute  alkali  extract  is  concentrated  under 
diiTiinished  pressure  to  about  I5O  cc.,  then  transferred  to  a digestion  flask  with 
an  equal  volume  of  concentrated  hydrochloric  acid,  and  completely  hydrolyzed  by 
boiling  on  the  hot  plate  for  I5  to  £0  hours  under  a reflux  condenser.  The  com- 
pletely hydrolyzed  proteins  in  this  solution  are  treated  as  directed  below  in  (J). 

E.  DIGESTION  WITH  h.O  PER  CENT.  TRICHLORACETIC  ACID. 

NOTE:  - This  extraction  is  unnecessary  in  the  case  of  feeds  having 
little  or  no  starch  present. 

The  residue  insoluble  in  dilute  sodium  hydroxide  is  transferred  to  a. 
1500  cc.  round  bottom  digestion  flask  with  5^0  cc.  of  k.O  'pei*  cent,  trichlora.ee tic 
acid  and  placed  on  a stea^n  bath.  With  frequent  shaking  the  solution  is  allowed 
to  digest  until  it  is  apparent  (from  the  disappearance  of  the  milky  color)  that 
much  of  the  starch  has  been  dissolved.  This  usually  takes  about  1 hour.  The 
, solution  is  filtered  wiiile  hot  through  a Buchner  funnel  fitted  with  liard  paper. 

The  residue  is  washed  a few  times  with  boiling  v/ater  and  the  residue  again  treated 
with  cc.  of  2.0  per  cant,  trichloracetic  acid  for  aboiit  a half  hour  period. 

Tna  hot  solution  is  filtered  and  washed  as  before  and  if  the  starch  is  not  all 
removed  this  treatment  is  repeated.  The  residue  insoluble  in  hot  2.0  per  cent, 
trichloracetic  acid  is  treated  as  directed  below  in  (E)  and  the  extract  as  direct- 
ed below  in  (I). 

F.  EXTRACTION  WITH  c:0  PER  CENT.  HYDROCHLORIC  ACID. 

'ihe  residue  insoluble  in  2.0  per  cent,  trichloracetic  acid  is  trans- 
ferred to  a round  bottom  digestion  flask  with  25O  cc.  of  20  per  cent,  hydrochloric 
acid.  The  solution  is  heated  to  boiling  on  a wire  gauze  and  boiled  for  3 minutes. 


;f  ■ 1.'  r 


' *^0  r<  ■ 


,fr 


iIiCfc.l4  • 


u 

f, ' 


;i  <X".-  • .’i 


V-./. 


’ . '> 


:r'  .' -c-x:  ..  . ..  *_  ' T.Q't  6J.-  1 . ' ..;  nr  ’.  ..  . . 

■I?.  ;T  ."JJ.J'Iv)  L;  viv  Jf : 


. -r  t . A 


— Ir 


»'  -<6. 


•..r..y  j 


jr.- 


t . 


\ 0^  H?F‘  '.“ITt'A'  ..... 


.i  r'..'j:/Irn  ’ -sci  ?r'- 

-c  • tao;Jv  oc\ 


X J iL  x09  a « « 


V 


-ii5- 

cooled  to  room  temperature,  and  filtered  through  hard  paper  on  a Buchner  funiiel 
with  suction.  This  procedure  is  repeated  once.  The  residue  is  washed  e£  or  3 
times  with  dO  per  cent,  hydrochloric  acid  and  than  with  ami..onia-free  water  until 
practically  free  from  acid.  The  wasnings  with  water  are  kept  separate  from  the  20 
per  cent,  hydrochloric  acid  extract.  The  20  per  cent,  hydrochloric  acid  extract 
and  washings  are  transferred  to  a digestion  flask  with  a volu'ne  of  concentrated 
hydrochloric  acid  equal  to  the  volume  of  the  washings.  iJiie  proteins  in  this 
solution  are  completely  li^'-drolyzed  in  the  usual  manner.  The  completely  hydrolyzed 
prcteins  are  treated  as  directed  below  in  ( J) . The  residue  is  treated  as  directed 
below  in  (G) . 

G.  EXTRACTION  WITH  STRONG  SODIUIvl  HYDROXIDE. 

The  residue  insoluble  in  20  per  cent,  hydrochloric  acid  is  transferred 
back  to  the  original  ^00  cc.  centrifuge  bottle  with  ammonia-free  water.  The  so- 
lution is  centrifuged  until  the  solid  rraterial  forms  a compact  mass  and  the  super- 
natant liquid  decanted  into  a properly  labeled  flask.  The  residue  is  then  ex- 
tracted for  three  2k  hour  periods  V7ith  5O  cc.  portions  of  5 per  cent,  sodium  hy- 
droxide. After  each  extraction  period  the  sides  of  the  bottle  are  washed  dom 
with  a few  cc.  of  aimTiOnia-free  water,  the  solution  centrifuged  until  the  solid 
matter  forms  a compact  mass,  and  the  supernatant  liquid  decanted,  the  extracts 
being  added  to  the  wrater  which  had  been  used  to  make  the  transfer  to  the  centri- 
fuge. Tne  residue  is  washed  2 or  3 times  in  the  centrifuge  bottle  with  100  cc. 
portions  of  armionia-free  water  in  the  usual  manner.  After  each  extraction  the 
extracts  are  a-cidified  v/ith  hydrochloric  acid.  Ttie  extract  is  concentrated,  if 
necessary,  then  transferred  to  a digestion  flask  v/ith  an  equal  volume  of  concen- 
trated hydrochloric  acid,  and  completely  hydrolyzed  by  boiling  for  IR  to  20  hours 
under  a reflux  condenser  on  the  hot  plate.  The  completely  hydrolyzed  proteins 
in  this  solution  are  treated  as  directed  belov/  in  (J).  The  residue  insoluble  in 


r k 


in  strong  sodixun  hydroxide  is  transferred  to  a Kjeldahl  flask  and  total  nitrogen 
determii.ed. 

H.  THEATlIEl^  07<’  THE  1 PEH  CEHT.  TRICHLOHACETIC  ACID  EXTRACTS. 

The  1 per  cent,  triculoracetic  acid  extract  is  brought  to  boiling^ 
niade  distinctly  alkaline  with  sodium  hydroxide  solution,  then  acid  to  litrnus  with 
hydrochloric  acid.  Add  very  slowly  10  cc.  of  colloidal  iron^  and  boil  for  1 
minute.  Three  cc.  of  a solution  of  crystallized  magnesium  sulfa-te  (made  by  dis- 
solving crystallized  fiagnesi-arn  sulfate  in  an  equal  volume  of  v\’ater)  is  added  to 
coagulate  the  excess  of  colloidal  ferric  hydroxide,  and  the  solution  again  boiled 
for  1 minute.  The  solution  is  allowed  to  stand  until  the  precipitate  settles, 
then,  without  filtration,  bring  to  the  boiling  point  and  reprecipitate  just  as 
before.  Allow  the  precipitate  to  settle  and  reprecipitate  a third  time.  During 
the  boiling,  precipitation,  and  standing  the  solution  gradually  becomes  alkaline 
due  to  the  decomposition  of  the  trichloracetic  acid.  The  slight  acidity  is  main- 
tained by  the  occassional  addition  of  hydrochloric  acid.  After  the  precipitate 
has  settled  the  third  time  the  solution  is  filtered  through  ha,rd  paper  on  a 
Buchner  funnel  with  suction.  The  solution  should  be  poured  on  the  filter  so  as 
to  filter  practically  all  of  the  liquid  before  much  of  the  precipitate  is  poured 
on  to  the  filter.  The  precipitate  is  vra.shed  thoroughl2;'  with  hot  v/ater.  The  fil- 
trate is  now  reprecipitated  again  in  exactly  the  same  manner  as  described  above 
and  filtered  on  a Buchner  funnel  fitted  with  a fresh  hard  paper.  The  filtrate  is 
now  cooled,  made  up  to  cc.  (3OOO  cc . if  necessary)  and  total  nitrogen  de- 

termined in  three  cc.  portions.  Transfer  the  colloidal  iron  precipitates  to 
a digestion  flask  v/ith  20  per  cent,  hydrochloric  acid  and  completely  hydrolyze 
by  boiling  on  a hot  plate  under  reflux  condensers  for  I5  to  20  hours.  The  com- 
o 

Iron  Dialyzed  Merck,  contains  5 cent.  Fe^Og. 


•VdfV 


-47- 


pletelj'’  liydrolyzed  proteins  in  this  solution  are  treated  as  directed  below  in  (J). 

I.  TRE.miEi'JT  OF  THE  ci  PER  CEIIT.  iRICHLORACETIC  ACID  EXTPJICTS. 

The  <L  per  cent,  trichloracetic  acid  extract  is  concentrated  under 
diminished  pressure^  without  neutralizing,  to  about  175  then  transferrec 

to  a beaker  upon  which  a mark  has  been  placed  to  indicate  25O  cc.,  made  up  to  the 
mark,  heated  on  the  steam  bath  until  most  of  the  starch  has  dissolved,  and  H vol- 
umes of  95  cent,  alcohol  added.  The  solution  is  allov/ed  to  stand  until  the 
precipitate  settles  completely  (2  or  3 days),  is  then  filtered  on  a Buchner  funnel 
fitted  with  hard  paper  and  washed  with  alcohol  of  the  same  strength  (2  volumes 
of  95  cant,  alcohol:  1 volunne  of  water).  Fne  starch  precipitate  is  transferrec 
to  a Kjeldahl  flask  and  total  nitrogen  determined.  The  filtrate  is  concentrated 
repeatedly  under  diminished  pressure  until  all  the  alcohol  is  removed.  It  is  then 
transferred  to  a round  bottom  digestion  flask  with  an  equal  volume  of  concentrated 
hydrochloric  acid  and  completely  hydrolyzed  by  boiling  for  6 to  10  hours.  The 
hydrolyzed  proteins  in  this  solution  are  treated  as  described  below  in  (J). 

J.  DETERIcIIlIATIOH  OF  INSOLUBLE  HUI.IIN  I^^ITR0GE^T. 

The  completely  hydrolyzed  proteins  from  (D),  (F),  (G),  (H),  and  (I) 
above,  are  filtered  tlirougii  the  same  Buchner  funnel  and  washed  a few  times  with 
hot  water.  The  residue  is  then  transferred  back  to  a digestion  flask  with  0.1 
per  cent,  hydrochloric  acid  and  refluxed  two  or  three  hours.  The  solution  is 
filtered,  washed  with  hot  water,  and  the  residue  again  boiled  for  1 hour  with  0.1 
per  cent  hydrochloric  acid.  After  this  treatment  the  residue  is  washed  thoroughly 
with  hot  water  and  the  insoluble  hurain  nitrogen  determined  by  submitting  this 
residue  to  Kjeldahl  ana,lysis.  The  filtrate  and  washings  from  the  above  are  united 
and  axialyzed  according  to  the  Van  Slyke  method  as  described  in  Part  III  below. 


i 


5 

i 


I fe  i.  : I 


!' 


rti::- 


. . T ’■  ' . •*  *»  £ c.  j 

,.j 

■.  •.•  r.  i ■'  ::'  J.J  r:.0'_'  '."U.'O'i  - if.* rij  ' 


PART  III.  ANALYSIS  OF  THP  HYDROLYZED  PROTEIITS. 


DETERIvlINATlON  OF  AvLiOMA  (A/IID  NITROGEtl) . Concentrate  the  filtrate 
and  washings  from  the  determination  of  the  insoluble  hurnin  nitrogen  in  Claissen 
flasks  luider  diminished  pressure  until  all  the  hydrochloric  acid  possible  has 
been  driven  off.  Wash  down  the  sides  of  the  flask  with  ajuiionia-free  water  and 
concentrate  again  almost  to  drjaiess.  Transfer  to  a kOO  cc.  measuring  flask  with 
amiv.orua-free  vvater  and  make  up  to  the  mark.  Now  transfer  to  a clean  one-liter 
Claissen  flask  witn  100  cc.  of  95  per  cent,  alcohol.  For  the  determination  of 
the  ammonia  arrange  the  Claissen  flask,  and  an  ordira-ry  one-liter,  distilling 
flask  as  shown  by  Van  Slyke,  (o)>  Fig.  1.  Now  add  10  per  cent,  suspension  of 
calcium  hydrate  until  alkaline  and  then  add  kO  cc.  in  excess.  Tne  apparatus  is 
then  joined  together  as  shown  in  the  figure  and  evacuated  to  a pressure  of  ^0  mm. 
or  less.  The  amount  of  n/iO  acid  added  to  the  larger  receiving  flask  should  be 
bO  cc.  and  the  amount  added  in  the  trap  should  be  3O  cc.  The  Glaissen  flask  is 
then  placed  in  a bath  at  U5“5*^'^>  the  solution  boiled  for  a half-hour.  In 

case  distillation  starts  too  rapidly  a little  air  is  let  in  from  the  stop-cock 
in  the  neck  of  the  Claissen. 

hhen  the  distillation  is  firhshed,  the  flask  is  lifted  from  the  water 
bath,  and  the  vacuum  is  released  by  first  allowing  a very  small  stream  of  air  to 
enter  through  the  side  am  and  then  through  the  capillary  and  finally  opening 
the  stop-cock  in  the  side  arm.  The  n/iO  acid  from  the  receiving  flask  and  the 
small  guard  flask  is  washed  into  an  800  cc.  titration  bottle  and  titrated  bade 
with  n/i0  sodium  hydroxide  using  alizarine  sulforjate  as  indicator. 

SOLUBLE  HIIvlIN  NITROGEN.  Drring  the  distillation  Lmiiiediately  above  all 
the  black  coloring  matter  as  soluble  huirin  is  absorbed  by  the  undissolved  lime. 
Filter  through  hard  paper  on  3 ii'icn  Buchner  furmel  using  suction.  V/ash  with 
ammonia-free  water  and  transfer  the  insoluble  residue  to  a beaker  vvith  about  850 
cc . of  armonia-free  water.  With  frequent  stirring  heat  gently  for  about  I5  min- 


-49- 


utes.  Filter  and  wash  thoroughly  with  ammonia-free  water.  Repeat  this  treat- 
ment once.  The  total  nitrogen  determined  in  the  residue  is  coxisidered  to  be 
the  soluble  humin  nitrogen. 

PRECIPITATIOI'I  OF  THE  BASES  (CYSTIHE,  LYSI!TS,  ARGININE,  AND  HISTIDINE) . 
The  filtrate  from  the  soluble  humin  is  neutralized  with  hjrdrochloric  acid,  re- 
turned to  the  vacuum  distilling  flask,  and  concentrated  to  about  65  cc.  Wash 
the  concenti’ated  solution  into  an  accurately  calibrated  200  cc.  measuring  flask 
and  dilute  to  the  mark.  Mix  thoroughly.  Now,  by  means  of  a 100  cc.  pipette 
calibrated  to  deliver  exactly  100  cc.,  transfer  100  cc.  of  the  well  mixed  solu- 
tion to  a clean  flask  and  wash  out  txie  pipette  with  water,  back  into  the  200  cc. 
measuring  flask.  Add  IS  cc.  of  concentrated  hydrochloric  acid  to  each  100  cc. 
portion  and  shake.  Place  I5  gra.  of  purified  phospho tungstic  acid  in  each  of 
two  3OO  cc.  Erlexjmeyer  flasks  upon  which  marks  have  been  placed  to  indicate 
200  cc.  Dissolve  the  phospho  tungstic  acid  in  a sirall  volume  of  water.  Now  to 
each  flask  containirig  the  phosphotuiigstic  acid,  add,  very  slowly  with  constant 
shaking,  a 100  cc.  portion  of  the  acidified  hydrolyzed  protein  solution.  Each 
solution  is  diluted  with  water  up  to  200  cc.  and  heated  in  a water  bath  until 
the  precipitate  of  the  bases  is  nearly  or  quite  dissolved.  The  temperatui’e  of 
the  solutions  should  be  increased  very  gradually.  The  bases  reprecipi tate  on 
cooling  e.s  crystalline  or  granular  phospho  tungstates.  Hie  solutions  are  allowed 
to  stand  4h  hours  for  the  precipitates  to  form  for  in  less  time  the  precipita- 
tion of  histidine  may  be  incomplete.  The  precipitates  should  not  be  allowed  to 
stand  much  longer  than  4^  hours. 

WASHING  OF  THE  BASES.  It. is  necessary  that  the  phosphotungst ic  acid 
precipitate  shall.be  washed  entirely  free  of  the  mother  liquors  and  the  amino- 
acids  of  the  unprec ip i table  fraction.  It  is  also  necessary/  ths.t  as  small  an 
amount  of  the  washing  solution  as  possible  shall  be  used,  in  order  that  the 
precipitate,  which  is  slightly  soluble  in  the  washing  solution  shall  not  dissolv( 


f 


-50- 

tc  an  appreciable  extent  during  the  washing. 

The  phospho tungstic  precipitate  of  the  bases  is  filtered  and  washed 
in  the  following  rnani^er;  A hardened  filter  paper  is  cut  to  the  proper  size  to 
fit  accurately  as  usual  against  the  bottom  of  a,  ^ inch  Buchner  funnel.  T^e  pre- 
cipitate is  poured  onto  the  filter  thus  prepared  and  the  mother  liquors  are  dravm 
off  as  completely  as  possible  by  a steady  and  moderately  strong  suction^  the 
precipitate  being  pressed  down  by  a flattened  rod.  Do  not  allow  the  precipitate 
to  be  sucked  dry.  The  v/ashing  is  carried  out  v/ithout  at  any  time  releasing  the 
suction.  Successive  portions  of  10  cc.  of  a washing  solution^  containing  3.5 
per  cent,  hydrochloric  acid  and  2.5  P^r  cent,  phospho tungstic  acid,  are  poured 
onto  the  well  packed  precipitate,  which  is  stirred  up  with  each  portion  by  means 
of  a flat- tipped  rod,  so  tiiat  all  parts  of  the  precipitate  are  reached  and  all 
lumps  well  broken  up.  If  a steady  suction  is  maiiitained  all  the  time,  this  can 
be  done  without  danger  of  loosening  the  filter  from  the  floor  of  the  funnel.  The 
first  three  or  four  portions  of  the  washing  solution  are  used  to  dislodge  the 
last  granules  of  the  precipitate  from  the  flask  in  which  the  latter  are  formed. 
The  succeeding  portions  are  added  from  a pipette  in  a fine  stream.  In  case  the 
first  four  washings  leave  a few  granules  of  the  precipitate  still  in  the  flask, 
they  are  allowed  to  remain  there,  as  they  are  already  sufficiently  v/ashed  and  the 
subsequent  portions  of  the  washing  solution  used  to  wash  the  precipitate,  in  the 
me.nner  just  described. 

The  washing  solution  should  be  cooled  to  0^  before  it  is  poured  onto 
the  precipitate.  It  lias  been  found  that  the  solubijity  of  the  phospho  tungstates 
of  the  hexose  bases  is  only  about  one-fourth  as  great  at  as  it  is  at  room 
temperature,  consequently,  when  the  washing  solution  is  used  at  or  near  0^  the 
danger  of  dissolving  appreciable  amount®  of  the  precipitate  is  reduced  to  a min- 
imum. The  number  of  washings  necessary  in  each  case  to  free  the  phospho tungstic 
precipitate  completely  from  the  mother  liquors  is  determined,  according  to  the 


-^1- 

raethod  of  Tan  Slyke,  by  testing  the  successive  washings  for  calciuni.  It  has  been 
found  that  6 washings  with  10  cc.  portions  are  usually  sufficient  to  remove  the 
calcium. 

DECOI'^POSITION  OF  THE  PH)  SPHOTUHGSTIC  PRECIPITATE  BY  A MIXTHPJE  OF 
ETHER  AND  AWYL  ALCOHOL.  Hie  precipitate  of  basic  phospho tungstates  is  removed 
from  the  filter  by  a spatula  and  washing,  and  transferred  to  a half-liter  separa- 
tory funnel,  using  200  to  3OO  cc.  of  water  to  effect  the  transfer.  Vrnen  the  pre- 
cipitate has  been  removed  as  completely  as  possible  by  mechanical  means,  the  fil- 
ter paper  is  spread  out  on  the  bottom  of  a dish  and  v/ashed  Vvith  water  made  alkaline 
v/ith  a few  drops  of  sodium  hydroxide.  This  dissolves  the  portions  of  precipitate 
imbedded  in  the  fibers  of  the  filter  paper.  In  case  any  granules  of  the  precipi- 
tate have  remsiined  in  the  flask  in  which  the  precipitate  was  originally  formed, 
these  are  either  washed  or  dissolved  out.  These  alkaline  washings  should  be 
neutralized  v/ith  dilute  hjrdrochloric  acid  before  being  added  to  the  main  portion. 
Seven  and  one-half  cc.  of  concentrated  hydrochloric  acid  are  added,  and  the  m»ix- 
ture  is  snaken  with  1:1  amyl  alcohol-ether,  using  so  much  of  the  later  that  it 
all  floats  in  a layer  above  the  water  after  the  precipitate  has  gone  into  solu- 
tion. Usually  about  100  cc.  of  the  ether -amyl  alcohol  suffices,  and  one  or  two 
mirmites  shaking  results  in  complete  solution  of  the  precipitate.  If  too  little 
of  the  ether-amyl  alcohol  has  been  used,  some  of  it  will,  after  taking  up  phos- 
photungstic  acid,  sirk  as  an  oil  below  the  aqueous  phase.  In  case  this  happens, 
more  of  the  extracting  mixture  is  added,  until  all  floats  in  one  layer  at  the 
top. 

In  some  cases  the  aqueous  and  ether -amyl  alcohol  layers  do  not  sep- 
are.te  readily  v/ith  a clean  boundary  betv/een  them.  This  effect  is  due  to  the  pres- 
ence of  a slight  amount  of  humin  which  may  have  escaped  previous  adsorption  by 
calcium  nydrate.  In  this  case  the  unadsorbed  huinin  is  carried  do\m  by  the  basic 
phosphotungstates,  and  fouls  the  solution  when  their  precipitate  is  decomposed 


as  above  described.  In  order  to  clear  the  solution  up^  it  is  all,  without  sep- 
aration of  the  aqueous  and  ether -cuiiyl  alcohol  layers,  passed  through  a Buclaner 
funnel  v;ith  suction.  The  residue  is  washed,  first  with  amaonia-free  water  and 
than  with  the  aiayl  alcohol-ether  mixture,  using  no  mors  washing  solutions  than 
absolutely  necessary.  The  residue  together  with  the  filter  paper  is  transferred 
to  a Kjeldahl  flask  and  total  nitrogen  determined.  This  fraction  of  nitrogen  is 
referred  to  as  the  "unadsorbed  humin  nitrogen."  The  filtrate  is  returned  to  the 
separatory  funnel,  tne  two  layers  allowed  to  separate,  and  the  aqueous  layer 
drawn  off  into  a second  !half-liter  separatory  funnel.  The  aqueous  layer  is  then 
extracted  with  three  more  successive  portions  of  ether-amyl  alcohol,  using  each 
time  a volume  of  the  mixture  equal  to  about  one-fourth  the  volume  of  the  water 
solution  (about  ^0  cc.).  Fiiially  the  combined  ether-amyl  alcohol  extracts  are 
shaken  out  twice  with  water  to  remove  traces  of  bases  that  might  have  been  carried 
into  the  extract;  this  portion  of  water  is  then  shaken  once  or  twice  with  fresh 
arijj'l  alcohol -ether,  axid  combined  with  the  main  solution  of  the  bases.  Tae  latter 
should  be  free  of  phosphotungstic  acid  as  demonstrated  by  the  absence  of  a precip- 
itate when  a few  drops  are  added  to  a saturated  solution  of  barium  hj^drate  in  a 
snail  test-tube.  The  amyl  alcohol-ether  solution  is  transferred  to  a Xjeldahl 
flask,  a few  pieces  of  pumice  a^nd  a few  drops  of  sulfuric  acid  added;  the  ether 
is  driven  off  by  heating  on  the  steam  bath;  the  axryl  alcohol  is  distilled  off, 
recovering  the  alcohol;  and  then  the  total  rutrogen  determined  in  the  residue  in 
the  usual  manner.  The  nitrogen  in  this  fra-ction  is  referred  to  as  the  "nitrogen 
soluble  in  amyl  alcohol-ether  mixture."  The  solution  of  the  bases  is  now  concen- 
trated to  dryness  under  diminished  pressure  in  order  to  drive  off  the  free  hydro- 
chloric acid.  Add  a small  amount  of  ammonia-free  v;ater  to  the  residue  and  thor- 
oughly and  comipletely  loosen  the  particles  adhering  to  the  sides  of  the  flask  with 
a properly  bent  glass  rod.  Wash  off  the  glass  rod  and  ’wash  dovm  the  sides  of  the 
flask  v/ith  a small  amount  of  water.  Concentrate  to  about  10  cc.  and  then  transfer 


-Si 


I' 


; T'- 


•:  ^ c'.  * r.:'.?  Hz.! 

. .%  •av.". 

* i.i  -jM-ri':  , - 

'li  ft'tirjji*  t'.  .f  :w'w  i 

\ 

1 1 t Xi 


V • 


. - vf%j  r .i  i"  ;;cl: 

i'-  «f  lien  ^ 


; . ,.  •jl';-  . •‘■’.  w’'-  *?•■«  XlJu'n  ..'‘rf 


I 


_^3- 

ths  concentrated  solution  of  the  bases  to  a 100  cc.  measuring  flask^  by  means 
of  a bent  glass  rod  and  small  portions  of  araraonia-free  v^ater.  Ivlake  the  solution 
up  to  the  mark  and  mix  thoroughly.  If  a residue  settles  out  on  standing,  filter 
through  a small  dry  quantitative  filter  paper  into  a clean  dry  flask.  Disca.rd 
the  first  cc.  of  the  filtrate  and  use  the  reriiainder  for  the  various  determinatiom 
of  the  bases  below.  V/ash  the  residue  on  the  filter  paper  thoroughly  ;vith  ammonia- 
free  ^'sater  and  discard  the  washings.  The  thorou.^ly  v/ashed  residue,  together  witl 
the  filter  paper,  is  submitted  to  Kjeldahl  analysis  and  the  nitrogen  referred  to 
as  the  "nitrogen  in  residue  filtered  from  solution  of  the  bases." 

DETERIvIIl'ATIOiJ  OF  ARGIFIIJE.  Of  the  100  cc.  of  solution  containing 
the  bases,  two  25  cc.  portions  are  placed  in  5OO  cc.  short  necked  Kjeldahl  flasks 
v/hich  are  connected  with  upright  Liebig  condensers,  Cc^rryir^  at  the  top  Folin 
bulbs  connected  to  the  condensers  by  rubber  stoppers.  Fifteen  cc.  of  K/10  acid 
are  placed  in  each  of  the  Folin  bulbs.  Twenty  five  cc.  portions  of  50  P-h  cent, 
sodium  hj^'dr oxide  solution  and  severa-l  bits  of  porosis  plate  are  added  to  the 
Kjeldahl  flasks.  The  solutions  are  then  boiled  gently  on  wire  gauzes  for  ex- 
actly five  and  one-half  hours,  then  let  the  water  out  of  the  condensers  and  boil 
for  one-half  hour  longer.  A small  stream  of  p^urified  air  is  passed  through  the 
apparatus  during  the  entire  six  hours.  This  is  simply  done  by  passing  the  air 
from  compressed  a-ir  pipes  through  a purifying  train  and  admitting  it  to  the 
apparatus  by  means  of  a capillarj'-  placed  in  the  stopper  of  the  Kjeldahl  flasks 
and  reaching  to  the  bottom  of  the  flasks.  The  acid  in  the  Folin  bulbs  is  trans- 
ferred to  titration  bottles  with  neutral  water  and  titrated  back  with  k/iO  alka- 
li, using  alizarine  suiforiite  as  indicator. 

DETEEivillATION  OF  THE  iluIITO  KITHOGEiT  OF  lEE  BASES.  Two  5 cc.  portions 
of  the  solution  containing  the  bases  are  used  for  the  amino-acid  nitrogen  deter- 
rnii'^tion  which  is  run  in  the  usual  manner  in  the  Van  Sl^/ke  machine.  The  deter- 
mination is  continued  for  a laalf  hour  period.  A blsmik  deterrairiation  of  the 


reagents  aiust  be  run  for  the  same  length  of  time. 

DETERJ'/illJATION  OF  THE  TOTAL  iMITROGEN  OF  TEE  BASES.  Two  10  cc.  por- 
tions of  the  100  cc.  solution  containing  t^e  bases  are  used  for  the  total  nitro- 
gen determination.  The  digestion  wi  tii  sulfuric  acid  must  be  continued  for  six 
hours  after  the  solutions  becoiiie  clear. 

DETEEIvlINATION  OF  CYSTINE.  The  amount  of  cystine  present  in  the  bases 
is  obtained  from  the  content  of  the  solution  in  organic  sxilfur.  Take  1^  cc.  of 
each  of  the  solutions  containing  the  bases  for  the  determination  of  cystine. 

Transfer  the  measured  portion  of  the  solution  containing  the  bases  to 
fused  silica  dishes  of  7 to  10  cm.  diameter  and  add  10  cc.  of  Denis'  modifica- 
tion of  Benedict's  solution.  Add  about  0.1  gm.  of  pure  sucrose.  The  mixture 
is  concentrated  to  :?ryness  on  the  water  bath.  It  is  then  heated  over  a free 
flame,  gradually  increasing  the  temperature  until  the  mixture  is  thoroughl;^ 
charred.  Then  place  the  dish  in  a muffle  furnace  heated  to  a dull  red  heat  for 
10  to  15  minutes.  Allow  to  cool.  Add  10  cc.  of  10  per  cent,  hydrochloric  acid 
and  dissolve  the  contents  by  warming  and  stirring  for  3 to  5 minutes.  Although 
the  solution  may  appear  to  be  perfectly  clear,”  filter  thrcu.gh  a good  grade  of 
quantitative  filter  paper  and  wash  tlioroughly  with  hot  v®,ter.  Dilute  the  solution 
to  about  150  cc.  and  heat  bo  boiling.  In  order  to  make  sure  that  an  excess  of 
barium  chloride  is  present,  while  boiling,  add  dnop  by  drop,  10  cc.  of  a filtered 
10  per  cent,  solution  of  barium  chloride.  Allow  to  stand  'd  days  and  if  the  pre- 
cipitate is  perfectly  white,  filter  on  weighed  Gooch  crvicibles.  If  the  precipi- 
tate produced  by  barium  chloride  is  yellow  indicating  impurities  the  precipitate 
must  be  purified  by  the  use  of  sodium  carbonate  in  the  usual  manner.  If  this 
procedure  is  followed  carefully,  purification  of  the  barium  sulfs.te  precipitate 
is  rarely  necessary. 

The  barium  sulfate  is  washed  and  v/eighed  as  usual.  The  weight  of 
barium  sulfate  obtained  must  be  corrected  for  the  amount  of  sulfur  found  in  the 


r 


¥ 


}K 

If 


I, 

tj 


i' 


i 


1 


t 

r v^ : 1 


ff 


/ 


_pt;_ 

j 

reagents  by  blanl:  analyses.  Tiiat  is^  for  each  series  of  cystine  deterrairiations 
two  blarik  anedyses  on  the  reagents  are  run  at  the  same  time.  In  the  reagents 
used  by  Van  Slyka  the  correction  v/as  I.5  mgs.  of  barixjm  sulfate.  Reagents  re- 
quiring a much  larger  correction  should  not  be  usedj  as  the  cystine  present  of- 
ten yields  only  a few  milligrams  of  barium  sulfate.  It  ha.s  been  found  that^  by 
repui'ifying  the  sodium  chloride  used  in  Derds*  reagent  and  using  fused  silica 
dishes  instead  of  porcelain,  the  blanks  are  exceptionally  low  and  constant, 
usually  varying  between  0.]  and  O.9  mgs. 

DETSEivilllATlOlT  OF  THE  TOTAL  NITROGEII  U THE  FILTRATE  FROM  THE  BASES. 

To  the  combined  filtrate  and  w'ashings  from  the  phospho tungstate  precipitate  of 
the  bases,  5O  cent,  sodium  hydrate  solution  is  added  until  the  solution 
turns  slightly  turbid  by  precipitation  of  lime  and  it  is  almost  neutral  to  lit- 
mus paper.  In  some  cases  a slight  precipitate  is  formed  before  the  solution  is 
neutral  and  it  readily  redissclves  by  the  addition  of  a fe-.;  more  drops  of  alkali. 
In  adding  the  alkali,  it  is  essential  that  the  neutral  point  should  not  be 
passed  by  more  than  a few  drops,  as  otherwise  a precipitate,  vmich  usually  dis- 
solves readily  upon  the  addition  of  a little  auetic  acid,  may  form  which  will 
not  dissolve.  The  solution  is  placed  in  a Claissen  flask  a-nd  concentrated  under 
diminished  pressure  until  salts  begin  to  crystallize  out.  The  solution  is  then 
washed  into  a 200  cc.  measuring  flask  and  diluted  to  the  mark.  Allovi  the  solu- 
tion to  stand  over  night  and  if  a precipitate  settles  out  filter  the  solution 
through  a dry  filter  paper  into  a dry  flask.  Total  nitrogen  is  deterniined  in 
the  filtrate  in  three  25  cc.  portions.  In  each  determination  use  one  and  one- 
half  times  the  us\ial  amount  of  reagents  and  continue  the  digestion  for  6 hours 
after  the  solution  has  become  clear.  Under  these  conditions  the  phospho tungstic 
acid  does  not  interfere  at  all  with  the  accuracy  of  the  determination.  The 
residue  which  was  filtered  from  the  solution  of  the  filtrate  from  the  bases  is 


washed  thorouglily  with  v/ater  and  su'Dinitted  to  Kjeldahl  ar^..lysis.  The  nitrogen  in 


• ^ . i i'*c. , . V. '.'  i^ir.:  ■ 


la,  ^-|  ^1  Mil  ’i'l 

irr.'S‘frf>  t9ca:«^lfum||P 


' iH  » ■ 


r 


-56- 

this  fraction^  which  lias  always  been  found  to  be  exceedingly  smll,  is  referred 
to  as  the  ’'nitrogen  in  the  residue  filtered  from  the  solution  of  the  filtrate 
from  the  bases.’' 

DETEExMIMTIOlI  OF  THE  iUyllUO  NITHOGEil  IN  THE  FILTRATE  FROM  THE  BASES. 

Tan  cc.  portions  of  the  filtrate  from  the  bases  are  used  for  the  ai:iino-acid 
determinations,  which  are  run  in  the  usual  manner  for  six  minutes  using  the 
large  burette. 

CALCULATION  OF  ARGININE,  CYSTINE,  HISTIDINi:,  LYSIIE  NITROGSI'J,  AND  THE 
NON-AMINO -AC  ID  NITROGEN  IN  THE  FILTRi-TE  FROM  'THE  BASES.  All  determinations  are 
expressed  in  percentage  of  the  total  nitrogen  of  the  feed  and  in  percentage  of 
the  feed.  The  determinations  on  the  solution  of  the  bases  and  on  the  solution 
of  the  filtrate  from  the  bases  are  corrected  for  the  solubility  of  the  bases 
accordirjg  to  the  tables  of  Van  Slyke.  It  is  to  be  remembered  tliat  the  hydrols’-sed 
proteins  from  an  entire  sample  were  made  up  to  200  cc.  and  two  100  cc,  portions 
taken  for  precipitation  which  was  carried  out,  as  far  a-s  quantities  of  reagents 
and  volumes  are  concerned,  according  to  the  method  of  Van  Slyke.  The  standard 
acid,  while  referred  to  as  n/10,  is  in  realitj^  sliglitl*/  less  than  that.  One  cc. 
of  the  acid  is  equivalent  to  0.0014  grams  of  nitrogen. 

(a)  ARGININE  NITROGEN.  The  grams  of  uncorrectad  arginine  nitrogen 
in  tlie  portion  of  the  sample  taken  for  precipitation  of  the  bases  is  equal  to 
the  cc.  of  acid  neutralized  multiplied  by  0.0014>  multiplied  by  2 (sines  only 
half  of  the  nitrogen  of  arginine  is  determined  as  ammonia),  multiplied  by  IOO/25 
(since  25  cc.  portion  of  the  100  cc.  solution  of  the  bases  \<.'ere  taken  for  argin- 
ine determination).  Fne  grams  of  corrected  arginine  nitrogen  in  an  entire  sample 
is  equal  to  the  grams  0 f uncorrected  arginine  rdtregen  obtained  above  plus  O.OO32 
.(the  solubility  correction  for  arginine)  multiplied  by  2OO/1OO  (since  half  of  the 
t:00  cc.  solution  of  the  hydrolyzed  proteins  of  the  whole  sample  was  taken  for 
precipitation  v/ith  phospho tungstic  acid). 


r V iT*/> - ■'  ,TV  r.'  .,»•  .f-'V.fv  .;• 


t.  - 1 

r>WJ,(^.:  ^ • Kj 


W " ■-  ^'i 


L_fe.  ,t>z?i'f-,f  •'?  U;h'l»y^;;i^'^j^4.,  ••  vM(». ». ,:iv 9|§’ 


v-4 


i*V.  \l  * 
' I . ' 


* -\ 


• ai04 

».i 


■,»r 


■ > «vV  ^ m:  - v^’'^;. . <y  ^^j,™ 

i : ■:  iV.4  HfT  \'fr 

»-.  ■■>•  *'  •■•■'»*  ; ■'  /jutL>rr  ■■  ■'•'•  '’’^•'l^M  '"  ‘ ' HP''''''*  ' iJ 


'■  ■ . / ■.  . ■ :• 


■** . 


W !}’.j  ..  '■  >.;>  ■t;l' V-Ttfrl^vUC*.  j}j,1 

^'.r;  •;’\  ' ’•’'«  “ v‘  "''■*;  ■*’’■  • . ' . * ■■ . '^vJFSf?? 

X'-i '!»  '- »-0>- ..4h 

j\T .n  ct>  '■  ' ' .J  '•'/' '‘•'■'r '■  . '^k*',, 


[U-  o.<  <V<  4 1 .c^.  ..•  • U;.iS 


,v>-  .‘‘•••■i  <v4'  •?'ji^)8f0|jB'  -'v  Jk5^  •■  IP 


iv  '>^  p 'i>,v  ,;'■; « ..'  ■' . l'i>  • :9 1 * < <4  .*1  •.  v*\44' 

-f*'  .t  ■ , . V ' ■£.  ■ r . » ' •^*  ’ ' 


' ■I'*  t .-..  ■*; 


,x  < »."..v  ..-•  -.n  •»  £ , 


'I'fWKiT^ 

' 'iv' , ^*1 . 


i)U(ij?pi‘ 


nA-  ?'3fi‘/,i..«tt,  -S&'i’i  -t:  ;<! 


L--.^'  ’ ’ *£‘‘  ‘ ' '•'•(<■■'■*'''  ''^'.  '■  2 ^ 

• .’  ■ ‘ ■ *..  . ' ■'  ■"''  ' ' k % .-.tfi  t ; ■ ; ;.j|)l  ‘ ^v]. 


'»S.''"i,'._'i«..f5 


j,  ,■,!■•  j ■ . . 'll  -.  ’ V ; ' I ’ !?■  ^ 2 ''  ' ■?  '^'  * ‘ 

-i  . i'  '•  .•  ^*1.10;:^, . \^h  . K * ^ '•  a*  ■ I’d  ■■) :' 

Si..;  :c# . ^vr-;-'.'^,  'f.  >J''  ..  ••  > ; .V-  ■ • y H ' vi  •'•  -5  . / y 


-•■v«'  — t 


,i  •'•■  •./  'rr‘‘  » uN«V  ’'’5<  AT-* ' '<  1 ^<>1 

ji.^.ilj.|i^  , -jQy-  -■--■-•fiiiriilf't^-i--,  [ . II '■  - 1 n HU...  I.  l■^^ll  ]ii- 


“57- 


(b)  CYS'i'I:\iE  i'JlTKuGE-v.  las  gx'cuiis  oi  uncorrected  cystine  m trogen  in 
t.ie  portion  of  tne  solutiox^  talien  for  precipitation  is  eq-ual  to  tne  grsms  of 
bariuii  sulfate  multiplied  by  0.0b0ou9,  multiuixei  by  1UO/15  (since  cc . of  tne 
lub  cc.  solution  of  tne  bases  were  taken  for  cj’'stins  det eriiunation) . rhe  grams 
Oi  corrected  cystine  nitrogen  in  an  exitire  saxxxple  is  eo;ucl  to  tne  grsans  of  un- 
corrected  cystine  nitrogen  obtained  above  plus  U.UU<io  (tne  solubility  cori  action 
for  cystine)  multiplied  by  cUO/lOO. 

(c)  HISTiDIi'.E  i'ilTJiOGK'i . fbie  non-amino  nitrogen  of  me  bases  comes 
from  tiie  argiuinej  wnicn  contains  txiree-fourtns  of  its  nitrogen  in  a foriu  wnicn 
does  not  react  witn  nitrous  acid,  and  from  tne  nistiaine,  wnicn  contains  two- 
thirds  of  its  nitrogen  in  non-amino  form,  rherefore  to  calculate  the  histidine 
nitrogen,  subtract  thre --fo-irtns  of  the  uncorrected  arginine  nitrogen  in  the  por- 
tioxi  taken  for  phospho tungstic  acid  precipitation  from  the  total  non-amino  rxitro- 
gen  (difference  betvxeen  total  nitrogen  and  amino  nitrogen),  axxd  multiply  the  dif- 
ference by  3/2,  or 

Histidixie  N = 3/2  (non-amino  N - 3/4  arginine  N) 

= 1.5  non-amino  IJ  - I.I25  arginine  N 

This  gives  the  gra-ms  of  uncorrected  histidine  nitrogen  in  the  portion  taken  for 
phospho tungstic  acid  precipitation.  The  grams  of  corrected  histidine  nitrogen 
in  an  entire  sample  is  erual  to  the  grams  of  uncorrected  histidine  nitrogen  ob- 
tained above  plus  0.003d,  multiplied  by  2OO/1OO. 

(d)  JLYSli'^E  hli’HOGEi!.  Tne  grams  of  unccrrected  lysine  nitrogen  in  the 
portion  taken  for  phospho  tungstic  acid  precipitation  is  e-iual  to  the  difference 
between  the  grcmxs  of  total  nitrogen  a.nd  the  sum  of  the  arginine,  histidixie,  and 
cystine  nitrogen,  ike  grams  of  corrected  lysine  nitrogen  in  an  entire  sample 

is  equal  to  the  grams  of  unccrrected  lysine  nitrogen  obtained  above  plus  O.OOOp 
(the  solubility  correction  for  lysine),  nniltiplied  by  2OO/1OO. 

(e)  hm-AidIh'O  NITlKOGElT  If)  THE  FILTEATE  FROM  THE  BASES.  The  grams  of 
uxicorrected  non-amino  nitrogen  in  the  filtrate  from  the  bases  in  the  portion 


y .;  ■•  ..r  ',1^*  .y',”  ,fH.T^ 

'■,  T ■■  • 


V. 


■r^^-  'i 


-''y'r  S'-  '■  'i  ^'< 


• ’<■  ■,•;.;■  -iJ"  ?j(tti 


;S  V \‘ 


{V,  . .;  ...  .; ■•'  yM,^ 


-A..  • 


.-.  i 


. * ■'  * J . V 


• I , » w . /i'  •«.  j I . . \ 

>»'j;**’.  ' ifri.  •'  .V  ♦'  A ' I ^ 


y., , . 


'f  VTK  »? 


S’ 


> , f , \ ■ ’ * 

■,%^aiP>.'-/6^:  ‘ f • ‘j  a';  . , ^ 


It 


• (J  ■'  ' ■ '•  -'y.?*  ■'*^';  s^^.. 


..  «W'M  • • 1 — ,;  . H ’.'.  "'y,  ■ 

'v*^/S^^v'"w',..  .’«  -•  s •’ f '■  i7  jS'.^ '/■  K** i 


. a 


.,.#■•<  t>.  f ,.'  -.  V' 

i' " ^.' - -.v/  V ■ < /*->i,%rt ^ 
^'  • ‘ ■ ' ,.  ■’..  \-H 


> r 


. •'  in-^v  * ) vi  '?.-:  .7> ,,  1 


1^,  yt  - 


i of 

^'.'yV.  v>’7y; 


' ■ ''  f ''.’  i 'i,i  ■ . 


s6n  ititf i ■ ; T/ftiJ  3^  >■  ^ 


V»-  /'•"•  .’  , '-  ■ i •■•-'.  , t'  y ....  ■’• 

7’'v  ■'•'  ,4- «'».'.x/ .4  y?,<  ,%.: f . - s 4>  ..4sji,ik^ 

’ J'-"  - '‘V'  V ”,  '•-«■■...■  \ 'i y"'  ^ •'  , ;■■  ■ ..kiJS’Viil?,. ' 

i,  ■•  ■••'  . • .‘  ' ’.•  . •.'■•Je..  . • •,.’  !’V'<  ’ «A’---‘v  V'  ’. .; 


L>VK^  .Jt'-'-^’'.  .•vv-.'fr’  ■■  .3V- ■ «^ .*1,  . '«.'J.  ■■'■'i' ' ' ‘ /MWBftT.  '' 

.:.,yr,#>- ; ' . iftiw*:. .a LT?b(j  *rii  #>(•*»  ^-.  . .-, - : 4*, 'BBMRT  . 


f 


. y-'.ii.  •'•  • ■■•■■V'-'''*V^.\S;.  .j.  : ■•‘^^'•'■'it{<'4'S  ;,iV."  ••’*:' ' '•'ST' >•••.! 


f Jli/  ti.  lA 


taken  for  precipitation  v/ith  phospho tungstic  acid  is  equal  to  the  difference 
between  the  grams  of  uncorrected  total  nitrogen  and  the  uncorrected  amino  nitro- 
gen in  the  filtrate  fran  the  bases.  The  grains  of  corrected  non-amino  nitrogen 
in  the  filtrate  from  the  bases  in  an  entire  sample  is  equal  to  the  grams  of  un- 
corrected  non-amino  nitrogen  obtained  above  minus  0.0049  (the  solubility  correc- 
tion), multiplied  by  SOO/lOO. 

PURITY  OF  REAGEilTS.  Since  some  of  the  calculations  are  based  on  dif- 
ferences between  determinations,  it  is  imperative  that  the  latter  shall  be  as 
accurate  as  possible.  Every  reagent  used  for  any  determination  involved  is 
tested  by  blank  analysis.  Blank  determinations  are  always  run  at  the  same  time 
all  amino-acid,  sulfur,  and  total  nitrogen  determinations  are  made  and  suite.ble 
corrections  made  for  the  reagents  used. 

Som.e  reagents  should  be  repurified.  The  sodium  chloride  and  the 
sugc,r  used  in  the  determination  of  sialfur  are  carefully  repurified.  The  phos- 
photungstic  acid  used  for  the  precipitation  and  washing  of  the  bases  is  repuri- 
fied according  to  the  method  of  Winterstein  (24)  and  the  tec'hnique  found  most 
suitable  for  this  purif ica,tion  is  as  follows;  I5  grams  of  phospho tungstic  acid, 
50  cc.  of  0.2  per  cent,  hydrochloric  acid,  and  9O  cc.  of  ether  are  placed  in  a 
separatory  funnel  and  shaken  until  the  solid  phospho  tungstic  acid  ]aas  completely 
dissolved.  On  standing  about  3 minutes  three  layers  separate.  The  clear  lower 
layer,  consisting  of  phospho tungstic  acid  in  ether  is  carefully  drawn  off  into 
a,  casserole  or  evaporating  dish.  Add  a drop  or  tv/o  of  concentrated  hydrocriloric 
acid  to  the  contents  of  the  funnel,  in  order  to  break  up  any  emulsion  that  had 
not  been  completely  broken  up  by  the  previous  treatment,  and  shake.  Allow  to 
stand  and  again  v/ithdraw  the  ether-phospho tungstic  acid  layer,  if  one  forms, 
into  the  casserole  or  evaporating  dish.  Repeat  this  procedure  until  no  more  of 
this  layer  is  obtained.  The  reinaining  two  lajrers  in  the  separatory  funnel  may 
be  used  for  the  purification  of  a second  I5  gram  portion  of  the  phospho tungstic 


-59- 


acid. 

The  ethereal  solution  is  allowed  to  evaporate  on  the  steaia  bath  at  a 
low  temperature  until  a thick  syrup  remains.  Then  set  avi/ay  from  the  heat  and 
direct  sunlight  and  allow  the  remaining  ether  to  evaporate  spontaneously  when 
crystals  of  purified  phcsphotungstic  acid  are  obtained,  iphe  latter  process  may 
be  hastened  by  placing  the  evaporating  dish  in  front  of  an  electric  fan  and 
blowing  a current  of  air  over  the  dish.  The  yield  is  about  6^  per  cent.  All 
the  liquors  remaining  from  the  purification  should  be  ^ved  and  the  phosplio tungs- 
tic acid  which  they  contain  recovered  later. 

VII.  DSTSRIvlIITATION  OF  THE  AIvlINO-ACIDS  OF  OATS,  COEIT,  COTTONSEED 

IvlEAL  AITD  ALFfJ.FA. 

By  the  detailed  method  outlined  above  in  section  VI,  6 samples  of 
oats,  6 of  corn,  8 of  cottonseed  meal,  and  U of  alfalfa  were  analyzed.  The  re- 
sults are  given  in  Table  I,  in  which  the  results  are  expressed  in  percentage  of 
the  total  nitrogen  of  the  feed,  and  in  Table  II,  in  which  the  results  are  ex- 
pressed in  percentage  of  the  feed.  The  oats  contained  l.oHO  per  cent,  nitrogen, 
the  corn  l.UOyU  per  cent.,  the  cottonseed  meal  6.79S  per  cent.,  and  the  alfalfa 
2.b2S  per  cent.  The. weights  of  feeds  taken  for  each  of  the  various  analyses 
were  60  grair4S  in  case  of  oats  and  corn,  3O  grams  in  case  of  alfalfa,  and  I5 
grams  in  cas^  of  cottonseed  meal.  Tne  oats  and  corn  v;ere  ground  so  as  to  pass 
through  an  80  mesh  sieve,  the  alfalfa  through  a 60  mesh  sieve,  and  the  cottonseed 
meal  through  a UO  mesh  sieve. 

A brief  examination  of  Tables  I and  II  will  show  that  all  solutions, 
residues,  precipitates,  and  other  fractions,  obtained  in  the  preparation  of  the 
hydrolyzed  protein  solution  and  in  the  subsequent  Van  Slyka  analysis  of  that  sol- 
ution, were  analyzed  for  their  nitrogen  content.  In  other  words,  no  fraction, 
v/hich  might  in  any  way  contain  any  portion  of  the  origira,!  sample  of  feed  taken 


X\> 


* ‘•''•‘i  ■» 


, '.t-/ 

iU-t 


I 


' 


-t 


■I 


J 


r 


L 


..'•a 


iiJlikni 


',  ■ > 


> 


' 'T'i.'*  *V^  ' 


-60- 


<«5 


TABLE  I. 


B 


DISTRIBU'ilOIT  OF  THE  ITITROOEiN  OF  OATS,  C0E1\',  COTTOIJSEED 
imj.  AlsT)  ALFALFA. 

(Expressed  as  Percentage  of  Total  Nitrogen'! 


D 


E 


H 


Nonprotein  Nitrogen 

Results  of  the 

-1 

Solu- 

Solu- 

In  fil- 

Total 

Insol- 

Solu- 

Acid 

Argin- 

Cys- 

Histi- 

— 

ble 

ble  in 

trate 

nonpro- 

uble 

ble  hu- 

amide 

ine 

tine 

dine 

in 

alco- 

from 

tein 

hmin 

min  nit- 

nitro- 

nitro- 

nitro- 

ni  tro- 

ether 

hoi 

colloid- 

nitro- 

nitro- 

rogen 

gen^ 

gen^ 

gen^ 

gen^ 

al  iron 

ge  n 

gen 

— — 

0.655 

1.172 

11.522 

13.3^9 

2.IS73 

2.783 

ll.Olb 

11.427 

0.961 

6.504 

r 2 

0.597 

1.163 

11,126 

12.386 

2.968 

3.^31 

11.033 

11.652 

1.008 

5.651 

s 

0.553 

1.041 

11.766 

13.360 

3.481 

1.498 

11 . 780 

11.888 

0.976 

5.543 

>1 

0.555 

1.375 

11.140 

13.070 

2.393 

1.71^8 

11 . 566 

11.474 

0.951 

5.942 

18 

0.513 

1.307 

10.360 

12.180 

2.611 

2.914 

11.350 

11.892 

0.894 

5.840 

1 

0.54U 

1.294 

10.360 

12.69s 

3.255 

2.926 

11.73s 

11.554 

0.876 

5.283 

7 

Aver.  0.569 

1.225 

11.129 

12.926 

3.013 

2.516 

11.422 

11.647 

0.944 

5.796 

3 

0,050 

0.997 

9.311 

10.35s 

1.571 

1.375 

11,729 

8.620 

1.099 

r5.2i*5 

i8 

0.792 

2.341 

8.093 

11.226 

1.796 

2.602 

12.265 

8.868 

1.165 

5»  460 

)8 

0,65a 

2.369 

3.395 

11.422 

1.499 

2.284 

12.225 

8.867 

1.186 

4.906 

)0 

O.cil^O 

0.239 

6.336 

7.315 

0.702 

2.685 

11.241 

8.782 

0.925 

4.748 

)8 

o.Ocia 

O.3O5 

7.316 

8.149 

0.758 

2.596 

12.218 

8.762 

0.935 

3.928 

)3 

0.139 

1.953 

3.356 

10.503 

1.084 

2.27s 

9.3331 

8. 451 

1.071 

4.702 

)2 

Aver.  0.3ci6 

1.363 

8.135 

9.329 

1.235 

3.303 

11.936 

8.725 

1.072 

4.832 

.0 

"T 

O.ORl 

0.570 

^.943 

5.534 

2.609 

3-462 

9.455 

18. 672 

0.961 

5.436 

;8 

0,039 

O.6I8 

4.870 

5-577 

2.609 

5.117 

9.689 

19.050 

0.902 

0.330 

)2 

0.202 

0.652 

5.053 

5.907 

2,492 

5.459 

9.929 

18.467 

1.068 

7.543 

'6 

0.109 

0.614 

5.531 

6.254 

2.623 

4.477 

8.892 

18.398 

1.123 

7.240 

10 

0.031 

O.5O0 

5.245 

5.S32 

2. 981 

2,415 

9.249 

17.520 

1.051 

8.534 

JO 

0.129 

0.489 

5.722 

6.340 

2,930 

2.650 

Q.3I8 

19.443 

0.Q48 

6.366 

i6 

0.031 

0.420 

6.097 

6.598 

2.763 

2-334 

9.002 

17.987 

0.707 

9.351 

J6 

0.0U6 

0.506 

7 .012 

7.564 

2.772 

2.746 

9.764 

20.102 

O.78I 

6.464 

?3 

Aver.  0,095 

0.51*7 

5-559 

b .201 

2.722 

3.582 

9.412 

18. 705 

0.943 

7.171 

f3 

— * 

0.577 

1.940 

16.466 

18.983 

3.632 

4.433 

6.943 

7.949 

0.924 

4.355 

+5 

0-577 

1.600 

16.301 

18.478 

3.6902 

3.512 

7.104 

8.064 

1.062 

3-655 

3l 

0.5^4 

1.9S3 

17.289 

19.801 

3.597 

5.132 

5.204 

7.523 

0.986 

3-782 

•‘L. 

0.522 

1.864 

16.712 

19.09s 

3.791 

4.796 

7.204 

8.446 

0.991 

3-931^ 

Aver.  0.550 

1.848 

16.692 

19.090 

3.690 

4.481 

7.364 

7.99b 

0.Q91 

^.9^1 

53 

^ Co 

rrected 

for  solubility  of  the  bases 

^ The  writer  is  indebted  chi 

sfly  to 

Nao  Uyei 

, assistant  chemist,  for  the 

following  arialyses  of  corn. 

The  write 

r is  inde 

bted  to 

W.  B.  Nevens  for 

the  following  analyses  of 

Dll- 

cottonseed  raea].. 

".f 

L^3LE  I.  riSTRI3UiI0ir  OF  THE  jlTROaEiN  OF  OATS,  COEN,  COTTONSEED 
;.3:/X  AlO)  ALFALFA 

(Eijjressed  as  Parcentage  of  Total  ITitrogen' 


A 

3 

C 

D 

E 

F 

G 

H 

I 

J 

7. 

L 

N 

0 

p 

0 

H i 

honprote 

in  Nitrogen 

Resiilts  of  the 

Van  SI 

yke  Analysis 

Nitrogen 

lost  in 

” Solu- 

ble 
in 

ether 

Solu- 
ble in 
alco- 
hol 

In  fil- 
trate 
from 
colloid- 
al iron 

Total 
nonpro- 
tein 
nitro- 
ge  n 

Insol- 
ubl  a 
hum  in 
nitro- 
gen 

Solu- 
ble hu- 
min  nit- 
rogen 

Acid 

amide 

nitro- 

gen^ 

Argin- 

ine 

nitro- 

gen* 

Cys- 

tine 

nitro- 

gen* 

Histi- 
dine 
ni  tro- 
gen* 

, Ly- 
sine 
k nit- 

K X 

rogen 

Amino 
acid  N 
in  fil- 
trate 
from 
bases^ 

Non-a- 
mino 
acid  N 
in  fil- 
trate 
from 
bases 

Total  non- 
protein + 
results  of 
Van  Slyke 
analysis 

N in  res- 
idue after 
treatment 
if.lth  strong 
NaOH 

In  alc- 
ohol ppt. 
of  hot  2 
pet . 

CCI3.CO2H 

extracts 

Unadsorbed 
hwflin  (fil- 
tered from 
sol.  during 
dacomp . of 
bases) 

In  axyl 
alcohol 
ether  | 
extract 

1 

1 

J 

OATS  (Contains  1. 

oSO  pet . N' 

0.655 

1.172 

11.522 

I3.349 

2.273 

2.723 

ll.Olo 

11.427 

0.961 

6.504 

£.122 

41.992 

4.102 

97-195 

0.120 

0.109 

0.552 

0.525 

0.^07 

1.163 

11.126 

12.Sdb 

2. 962 

3.231 

11.023 

11.652 

1.002 

5.651 

2.221 

41.921 

2.964 

96.245 

0.0'^5 

0.102 

0.235 

0.771 

0.553 

l.OUl 

11.766 

13.360 

3.1421 

1.492 

11.720 

11.222 

0.976 

5.543 

3.445 

42.174 

3.209 

97-954 

0.136 

0.142 

0 . 506 

0.759 

0.555 

1.375 

ll.lUO 

13.070 

2.293 

1.742 

11 . 566 

11.474 

0.951 

5.942 

; 2.326 

41.922 

4.922 

96.946 

0.165 

0.123 

0.251 

0.029 

0.513 

1.307 

10.360 

12.120 

2.611 

2.914 

11.350 

11.292 

0.2P4 

5.240 

3.426 

42.O06 

3.522 

96.751 

0.142 

0.153 

0.340 

0.755 

0.5LL 

1.29U 

10.S60 

12.69s 

3.255 

2.926 

11.73s 

11.554 

0.276 

5.223 

2.727 

42.622 

3.704 

97.503 

o.i6s 

0.123 

0.233 

0.732 

Aver.  0.569 

1.225 

11.129 

12.926 

3.013 

2.516 

11.422 

11.647 

0.944 

5.796 

2.241 

42.137 

3. 260 

97.100 

0.132 

0.127 

0.664 

0.746 

7 ...  s T 

u 

V 

In  resi- 

In  resi- 

Total 

To 

Total 

due  fil- 

due  fil- 

ni  tro- 

nitro- 

1 

tered 

tered 

gen 

gen  ac- 

from  so- 

from  so- 

lost 

counted 

1 

lution 

lution  of 

for 

1 

of  bases 

filtrate 

J.. 

from  btises 

0.363 

0.024 

1.699 

92.294 

— 

0-297 

0.033 

2.oqq 

36 . 344 

1 

O.OS3 

0.015 

1.707 

99.661 

0.141 

0.025 

2.234 

99.120 

0.191 

0.020 

1.607 

9S.362 

0.179 

0-033 

2.074 

99-577 

0.209 

0.025 

1.903 

99.004 

ALFALFA  (Contains  2.6dS  pet 


0.577 

1.940 

l6*4bo 

12.923 

3.622 

4.423 

6.943 

7.949 

0.924 

4.356 

4.334 

3S.349 

3.863 

93-866 

2.663 

Extrac- 

0.999 

0.609 

Not 

0.144 

4.415 

98.221 

0.577 

1.600 

16.301 

12.472 

3.6902 

3.512 

7.104 

8.064 

1.062 

3-655 

4.959 

37.681 

3.059 

91.264 

2.335 

tion 

1.110 

0.216 

de- 

0.639 

4.900 

96.164 

0.524 

1.922 

17.229 

19. 801 

3.597 

5.132 

2.204 

7.523 

0.926 

3-782 

4.002 

37.312 

1.196 

91.541 

2.930 

not 

1.274 

O.3OI 

term- 

0.604 

5.109 

96.650 

0.522 

1.264 

16.712 

19.098 

3-791 

4.796 

7.i!04 

3.446 

0.991 

3.931^ 

4.4342 

32.726 

1.927 

93-404 

2.146 

made 

1.261 

0.717 

ined 

0.311 

4.501 

97.905 

Aver.  0.550 

1.242 

16.692 

19.090 

4.481 

7.364 

7.996 

o.qqi 

3-931 

4.434 

32.032 

<i.5ll 

92.5<;0 

2.519 

1.161 

0.611 

4 

0.441 

4.732 

97-25<: 

m. 


0.050 

0.997 

9.311 

10.352 

1.571 

1.375 

11.729 

2.620 

1.099 

5.245 

2.424 

46.090 

5. 600 

94.111 

O.5051 

0.513 

0:930 

0.521 

0.129 

O.O65 

2.723 

96.234 

— 

0.792 

2.341 

8.093 

11.226 

1.796 

2.602 

12.265 

2.268 

1.165 

5.46b 

:2.41s 

46.790 

0.224^ 

93.474 

0.176 

0.163 

0.930 

0.527 

0.206 

1.4681 

3-U7O 

96.944 

0.652 

2.369 

8.395 

11,422 

1.499 

2.224 

12.225 

2.867 

1.186 

4.906 

I2.473 

45.059 

2.251 

90.172 

0.147 

0.335 

1.999 

0.515 

o.a30 

0.002 

3.234 

101.406 

0.240 

0.239 

6.836 

7.315 

0.702 

2.625 

ii.24i 

2.722 

0.925 

4.74s 

1.847 

47.750 

2.763 

94.758 

0.107 

0.265 

3-626 

0.372 

0.149 

0.133 

4.7I8 

99.470 

0.022 

O.3O5 

7-816 

2.149 

0.758 

2.596 

12.218 

2,762 

0.925 

3.922 

2.221 

47.962 

6.270 

94. 449 

0.113 

0.073 

2.791 

0.531 

0.164 

0.045 

3-717 

92.166 

0.129 

1.958 

2.356 

10.503 

1.024 

2.272 

9.8331 

8. 451 

1.071 

4.702 

1.214 

46 . 57  4 

q-599 

Q2.909 

0.137 

0.305 

5.250 

0.412 

0.209 

0.075 

6-994 

99.903 

Aver.  0.326 

1.368 

8.135 

9.229 

1.235 

2.303 

11.936 

8. 725 

1.072 

4.832 

2.200 

46.704 

7.216 

96.052 

0.136 

0.276 

2.698 

0.421 

0.191 

0.065 

3.247 

99-899 

COTTONSEED  MEAL^ 

(Contains  b 

• 796  pet.  N'l 

0.021 

0.570 

4.943 

5-534 

2.609 

'3.462 

^ 9.455 

12.672 

no.qpi 

5-486 

4.240 

39-981 

3-871 

93-671 

0.h20 

2.274 

0.920 

0.20b 

0.036 

3-7I6 

97.38? 

0,029 

O.6I8 

4.870 

5-577 

2.609 

5.117 

9.629 

19.050 

0,902 

6.330 

3.060 

40.539 

3.432 

96 . 3O5 

0.260 

1.562 

0.906 

0.215 

0.039 

2.922 

99.^87 

0.202 

0.652 

5.053 

5.907 

2.492 

5-459 

9.929 

18.467 

1,068 

7.543 

3-570 

38.852 

1.901 

95.122 

0.30a 

Extrac- 

1 .652 

0.722 

O.22O 

0.106 

3-O74 

99.262 

0.109 

0.614 

5-531 

0.254 

2.623 

4.477 

2.8q2 

IS. 398 

1.123 

7.240 

4.470 

39-828 

0.1611 

93-466 

0.233 

tion 

3.044 

1.135 

0.245 

0.130 

4.787 

98.253 

0.021 

5.245 

5.832 

2.921 

2.415 

9.249 

17.520 

1.051 

8.524 

4.462 

41.958 

2.621 

96.733 

0 . 4302 

not 

1.192 

0.691 

0.422 

0.150 

2.291 

99.624 

0.129 

0.489 

5.722 

6.340 

2.930 

2.650 

9.3I8 

19.443 

o.q48 

6.366 

4.998 

41.950 

2.290 

97.833 

0.625 

ifiade 

1.019 

O.7SO 

0.320 

0.096 

2.960 

100.193 

0.021 

0.420 

6.097 

6.592 

2.763 

2.334 

9.CO2 

17.987 

0.707 

9.351 

3.600 

39.204 

3.027 

94.633 

0.719 

0.772 

i.oa6 

0.096 

O.O05 

2.672 

97.311 

0.046 

O.5O6 

7.012 

7 - 564 

2.772 

2.746 

9.764 

20.102 

0.721 

6.464 

_ 5-874 

43-481 

3-454 

101.402 

0.529 

1.364 

1.068 

0.133 

0.068 

3.222 

105.624 

Aver.  C.O95 

0.547 

5-559 

6.201 

2.7H2 

3-582 

9.412 

12.705 

0.943 

7.171 

^ 4.209 

40.724 

2.874 

96.543 

0.430 

1.611 

0.922 

0.<;40 

0.026 

3.229 

99.832 

xui  ouxuuxxj.  ui  x,ne  o^ses 

The  writer  is  indebted  chiefly  to  Nao  Uyei,  assistant  chemist,  for  the 
^ following  arialyses  of  corn. 

The  writer  is  indebted  to  W,  B.  Nevens  for  the  following  analvsas  of 
cottonseed  maaj. 


Not  included  in  the  average 

2 Determination  lost;  average  result  substituted  to  make  up  total 


TABLE  II.  DISTRIBUTION  OF  THE  NITROGEN  OF  OATS,  COEN,  COTTONSEED 

MEAL  AND  ALFALFA 

(Expressed  as  Percentage  of  Feedingstuff ) 


A 

B 

C 

D 

E 

F 

G 

H 

I 

J 

Nonprotein 

Nitrogen 

Results  of  the 

Solu- 

'ble 

in 

ether 

Solu- 
ble in 
alco- 
hol 

In  fil- 
trate 
from 

colloid- 
al iron 

Total 

nonpro- 

tein 

nitro- 

gen 

Insol- 
uble 
hum  in 
nitro- 
gen 

Solu- 
ble hu- 
Viin  nit- 
rogen 

A-cid 

aiTjide 

nitro- 

gen^ 

Argin- 

ine 

ni  tro- 
gen 

Cys- 

tine 

nitro- 

gen^ 

Histi- 

dine 

nitre- 

/-r 

gen 

0.0110 

0.0197 

0.1936 

0.2242 

0.0482 

0.0467 

O.I85O 

0.1919 

0.0161 

0.1092 

0.0100 

0.0195 

O.IS69 

0.2164 

0.0498 

0.0542 

0.1862 

0.1957 

0.0169 

0.0949 

0.0093 

0.0175 

0.1977 

0.2244 

0.0584 

0.0251 

0.1979 

0.1997 

0.0164 

0.0931 

0.0093 

0.0231 

O.I672 

0.2195 

0.0466 

0.0293 

0.1943 

0.1917 

0.0159 

0.0998 

O.OOSo 

0.0220 

0.1740 

0.2046 

0.0436 

0.0469 

0.1906 

0.1997 

0.0150 

0.0981 

0.0091 

0.0217 

0.1624 

o.aX33 

0.0546 

0.0491 

0.197s 

0.1941 

0.0147 

0.0887 

Aver.  0.0096 

0.0206 

O.IS7O 

0.2170 

0.0506 

0.0422 

0.1916 

0.1956 

0.0158 

0.0973 

0.0007 

0.0140 

0.1310 

0.1456 

0.0221 

0.0194 

0.1651 

0.1213 

O.OIR5 

0.0738 

0.0111 

O.O33O 

0.1139 

O.I58O 

0.0253 

0.0366 

0.1726 

0.1246 

0.0164 

0,0 1 08 

0.0093 

0.0333 

0.1162 

0.1608 

0.0211 

0.0321 

0.1721 

0.1246 

0.0167 

0.0690 

O.OO3U 

0.0034 

0.0962 

O.IO3O 

0.0099 

O.O378 

0.1582 

0.1236 

0.0130 

0,06b8 

0.0004 

0.0043 

0.1100 

0.1147 

0.0107 

0.0365 

0.1720 

0.1233 

0.0139 

0.0553 

O.OOciy 

0.0276 

0.1176 

0.1472 

0.0153 

0.0321 

0.1334^ 

0.1189 

0.0151 

0.0662 

Aver.  0.0046 

0.0193 

0.1145 

0.1363 

0.0174 

0.0324 

0.1680 

0.1228 

0.0151 

0.0680 

0.0014 

0.0367 

0.3360 

0.3761 

0.1773 

o.a35£ 

0.6426 

1.2689 

0.0653 

0.3728 

0.0061 

0.0420 

0.3310 

0.3790 

0.1773 

0.3476 

0.6585 

1.2946 

0.0613 

O.43O2 

0.0137 

0.0443 

0.3434 

0.4014 

0.1694 

0.3710 

0.6746 

1.2550 

0.0726 

0.5126 

0.0075 

0.0417 

0.3459 

0.4250 

0.1783 

O.3O43 

0.6043 

1.2503 

0.0763 

0.4920 

0.0055 

0.0345 

0.3565 

0.3963 

0.2026 

0.1641 

0.6286 

1.1906 

0.0714 

0.5830 

0 . 0036 

0.0332 

0.3669 

0.4309 

0.1991 

0.1801 

0.6333 

1.3213 

0.0644 

0.4326 

0.0055 

0.0265 

0.4144 

0.4464 

0.1878 

O.I5S6 

0.6118 

1.2224 

0.0480 

0.6356 

0.0032 

0.0344 

0 . 47  95 

0.5140 

0.1884 

0.1866 

0.bb35 

1.3661 

O.O63I 

0.4393 

Aver.  0.0065 

0.0372 

0.3778 

0.4214 

O.I85O 

0.2434 

0.6396 

1.2712 

0.0641 

0.4^73 

0.0152 

0.0510 

0.4327 

0.4969 

0.096s 

0.1178 

0.1S25 

0.2089 

0.0243 

0.1145 

0.0152 

0.0420 

0.42o4 

0.4356 

0.09702 

0.0923 

0.1S67 

0.2009 

0.0279 

0.0961 

0.0136 

0.0522 

0.4544 

0.5204 

0.0945 

0.1349 

0.2156 

0.1977 

0.0259 

0.0994 

0.0137 

0.0490 

0.439^i 

0.5019 

0.0996 

0.1260 

0.1893 

0,2220 

0.0260 

0.103>' 

Aver.  0.0145 

0.0466 

0.4397 

0.5017 

0.0970 

0.1178 

0.1935 

0.2101 

0.0260 

0.1033 

^ Corrected  for 

solubility  of  the 

bases 

y The  wr 

iter  is 

indebted  chiefly  to  Nao  Uyei,  assistant  chemist; 

for  the 

following  analyses  of  corn 

The  vvTiter  is 

indebted  to  W.  B. 

Nevens 

for  the 

following  an^.lyses  of 

cotton- 

seed  meal. 

■ _p 

IA3LE  II. 


DISTRIBUTIO:;  OF  THE  NITROGEN  OF  OATS,  COEN,  COTTONSEED 
.'.SAL  AND  ALFALFA 

(Expressed  as  Percentage  of  Feedings tnff) 


G 


H 


H 


Solu- 

tle 

in 

ether 


Korjrotein 


SolU' 
tie  in 
alco- 
ol 


Nitrogen 


In  fil 
trate 
from 

colloid- 
al iron 


Total 

nonpro- 

tein 

nitro- 

gen 


Insol' 
uble 
hum  in 

nitro-  jrogen 
gen 


Solu- 
ble hu- 
r.in  nit- 


Aoid 

amide 


nitro-  nitro-  nitro 


Results  of  the  Van  SI  yVa  Arfl.lysis 


gen^ 


Total  non- 
protein  + 
results  of 
Van  Slyke 
analysis 


N in  res- 
idue after 
treatment 
vith  strong 
NaOH 


O.CllO 

O.OIQ7 

0.1936 

0.2242 

0.0432 

0.0467 

O.I85O 

0.1919 

0.0161 

0.1092 

0.0366 

0.7054 

O.O69O 

1.632s 

0.0020 

0.0018 

0.0094 

0.0088  1 

0.0100 

0.0195 

0.18o9 

0.2164 

0.0498 

0.0542 

O.I862 

0.1957 

0.0169 

0.0949 

0.0474 

0.7052 

0,0498 

1.6169 

0.0009 

0.0018 

0.0140 

0.0130  ' 

0.0093 

0.0175 

0.1977 

0.2244 

0.0584 

0.0251 

0.1979 

0.1997 

0.0164 

0.0931 

0.0578 

O.7O85 

0.0640 

1.6456 

0.0023 

0.0025 

0.0095 

0.0128  1 

0.0093 

0.0231 

0.1372 

0.2195 

0.0486 

0.0293 

0.1943 

0.1927 

0.0159 

0,0998 

0.0400 

0.7044 

O.OS'^S 

1.6287 

0.0028 

0.0021 

0.0143 

o.oi‘=6  ’ 

0.0080 

0.0220 

0.1740 

0.2046 

0.0438 

0.0439 

0.1906 

0.1997 

0.0150 

0.0981 

0.0575 

0.7067 

0.0602 

1.6255 

O.OO25 

0.0026 

0.0057 

0.0127  ' 

0.0091 

0.0217 

0.1624 

0.2133 

0.0546 

0.0491 

0.1972 

0.1941 

0,0147 

0.0887 

0.0468 

0.7071 

0.06^2 

1.6380 

0.0028 

0.0021 

0.0140 

0.0124  1 

Aver.  0.0096 

0.0206 

O.I87O 

0.2170 

0.0506 

0.0422 

0.1913 

0.1956 

0.0158 

0.0973 

0.0477 

0.7079 

0.0648 

1.6312 

0.0022 

0.0021 

0,0112 

0.0125 

OATS  (Contains  l.bbO  pet.  N) 


In  alc- 
ohol ppt. 
of  hot  '<L 
pet. 

CClp.COgH 

extracts 


R 

Nitrogen  lost  in' 


Unadsorbed 
humin  (fil- 
tered from 
sol.  during 
deconp.  of 
Bases'! 


In  a.T.yl 
alcohol 
ether 
extract 


0.0007 

0.0111 

0.0093 

0.0034 

0.0004 

0.00^7 

Aver.  0.0046 


0.0140 

O.O33O 

0.0333 

0.0034 

0.0043 

0.0k7o 

0.0193 


0.1310 

0.1139 

O.llBH 

0.0962 

0.1100 

0.1176 

0.1145 


0.145s 

O.I58O 

O.loOS 

O.IO3O 

0.1147 

0.147s 

0.1383 


0.0^21 

0.0253 

o.oai 

0.0099 

0.0107 

0.0153 


0.0174  0.0324 


0.0194 

0.0366 

0.0321 

0.0378 

0.0365 

0.0321 


0.1651 

0.1726 

0.1721 

0.1582 

0.1720 

0.1334^0 


0.1680  0.1228 


1213 

124H 

1243 

1236 

1233 

1189 


0.011^5 

0.0164 

0.0167 

0.013O 

0.0139 

0.0151 

0.0151 


0.07^ 
0 . 07  6s 
0.0690 
0.0668 
0.0553 
0.0662 


0.0680  0.0310 


[0.0341 

0.0340 

0.0348 

0.0260 

0.0313 

0.0255 


CORi#  (Contains  1.4074  Pct.  N) 


0.6487 

0.6585 

0.6342 

0.67<i0 

6750 

6555 


0.6573 


0.0788 

0.0124 

0.1161 

0.1233 

0.0967 

0.0929 

0.1016 


1.3845 

1.3156 

1.3817 

1.3336 

1.3293 

1.3076 

1.3518 


0.0014 

0.0061 

0.0137 

0.0075 

0.0055 

0.0086 

0.0055 

0.0032 

Aver.  0.0065 


0.0337 

0.0420 

0.0443 

0.0417 

0.0345 

0.0332 

0.0265 

0.0344 

0.037. 


0.3360 

0.3310 

0.3434 

0.3459 

0.3565 

0.3689 

0.4144 

0.4765 

0.3778 


0.3761 

0.3790 

0.4014 

0.4250 

0.3963 

0.4309 

0.4484 

0.5140 

0.4214 


0.1773 

0.1773 

0.1694 

0.1733 

0.2026 

0.1991 

0.1878 

0.1884 

O.I85O 


0.2352 

0.3473 

0.3710 

0.3043 

0.1641 

0.1801 

0.1586 

0.1866 

0.2434 


0.6426 
0.6535 
0.6743  _ 
0.6043  ll 
0 . 6286 
0.6333 

0.6113 

0.6635 

0.6396 


.2639 
.2946 
,2550 
,2503 
.1906 
■381 3 
,2224 
.3661 

,2712 


0053 
061 3 
0726 
0763 
0714 
0644 
0430 
0631 


0.0641 


0.3728 

0.4302 

0.5126 

0.4920 

0.5630 

0.4326 

0.6356 

0.4393 

0.4373 


0.2832 

O.2O5O 

0.2426 

0.3038 

0.3032 

0.3399 

0.2447 

0.2905 

0.2860 


COTTONSEED  MEAL^  (Contains 


00711 
0.0025 
0.0021 
0.0015 
0.0016 
0.0019 

0.0019 


0.0072 

0.0023 

0.0047 

0.0037 

0.0010 

0.0043 


0.0039 


2.7170 
2.7550 
2.6404 
2.7067 
2. 3514 
2.8509 
2 . 6643 
2.9550 

.7676 


0.2223 

0.2332 

0.1292 

0.0109: 

0.1822 

0.1556 

0.2098 

0.2347 

0.1953 


6.3659 
6. 4543 
6.4690 
6.5319 
6.5740 
6.6080 
6.4313 
6.3913 

6.5611 


0.0152 

0.0152 

0.0133 

0.0137 

Aver.  O.CI45 


0.0510 

0.0420 

0.0522 

0.0490 

0.0436 


0.4327 

0.4264 

0.4544 

0.4392 

0.4337 


0.4939 
0. 4356 
0.5204 
0.5019 

0.5017 


0.0968  b.1178 
O.O97O2  0.0923 


0.0945 
0.0996 


0.0970  0.1178 


0.1349 

0.1260 


0.1825 


0.2069 


O.IS67  p.20oq 
0.2156 

0.1893 


0.1935 


0.1977 

0.2220 

0.2101 


0.0243 

0.0279 

0.0259 

0.0260 

0.0260 


0.1145 

o.oqoi 

0.0994’ 

0.1033? 

0.1033 


* Corrected  for  solubility  of  the  bases 
y The  writer  is  indebted  chiefly  to  Nao  Uyei,  assistant  chemist,  for  the 
^ following  analyses  of  corn 

The  writer  is  indebted  to  1^.  B.  Nevens  for  the  following  ane.lyses  of  cotton' 


ALFALFA.  (Contains  2.628  Pct.  N) 


0.1139  1.007s 
O.I3O3  D.9902 
0.1053  D.9806 
0.116521.0193 

0.1165  0.9995 


0.1015 

0.0604 

0.0314 

O.O5O6 

0.0660 


2. 4668 
2.3934 
2.4057 
2.4547 

2.4314 


6.796  pct.  N^ 


0.0131 

0.0131 

0.0281 

0.0519 

0.0393 

0.0823 

O.O38O 


0.0073 

0.0074 

0.0072 

0.0052 

0.0075 

0.0059 

0.006s 


0.0150 

0.0177 

0.0205 

0.0158 

0.02922 

0.0465 

0.0439 

0.0400 

0.0292 


Extrac- 
tion not 
made 


0.1545 

0.1062 

0.1127 

0.2069 

0.0810 

0.0693 

0.0525 

0.0927 

0.1095 


0.0666 

0.0016 

0.0536 

0.0771 

0.0470 

0.0530 

0.0697 

0.0726 

0.0627 


0.0700 

0.0614 

0.0770 

0.0564 

0.0622 


Extrac- 
tion not 
made 


0.0263 

0.0292 

0.0335 

0.0331 

O.O3O5 


0.0160 

0.0214 

0.0079 

0.0188 

0.0161 


^ Not  included  in  the  average 

2 Determination  lost  - Average  result  substituted  to  ma’xe  up  total 


seed  meal . 


s 

T 

u 

V 

method  of  aralvsis 

Total 

In  resi- 

In  resi- 

Total 

Total 

due  fil- 

due  fil- 

nitro- 

nitro- 

tered 

tered 

gen 

^:en  ac- 

from  so- 

from  so- 

lost 

counted 

lution 

lution  of 

for 

of  bases 

filtrate 
from  bases 

O.OOol 

0.0004 

O.O285 

1.6614 

0.0050 

0.0006 

0.0352 

1.6521 

0.0014 

0.0003 

0.0236 

1.6743 

0.0024 

0.0004 

0.0375 

1 . 6662 

0.0032 

0.0003 

0.0270 

1.6525 

0.0030 

0.0006 

0.0343 

1.6729 

0,0035 

0.0004 

O.O3I9 

1 . 663*^ 

0.0027 

0.0009 

O.O3S3 

1.3628 

0.0029 

0.02071 

0.0438 

1.3644 

0,0032 

0.0001 

0.0455 

1.4271 

0.0021 

0.0019 

0.0663 

1.3999 

0.0023 

0.0006 

0.0523 

I.38I6 

0.0029 

0.0011 

0.0984 

1.40b0 

. 

0.0027 

0.0009 

0.0541 

I.406O 

0.0140 

0.0024 

0.2525 

6.6134 

0.0146 

0.0026 

0.2027 

6.7432 

0.0150 

0.0072 

0.2089 

6.6779 

0.0167 

0.0083 

0.3253 

6.6773 

0.0291 

0.0102 

0.1965 

6.7704 

0.0258 

0.0065 

0.2011 

6.8O91 

0.0065 

0.0044 

0.1820 

6.6132 

0.0090 

0.0046 

0.2190 

7.1102 

0.0163 

0.0058 

0.2235 

6.7346 

0.0038 

0.1160 

2.5828 

Not 

0.0168 

0.1288 

2.5272 

deter- 

0.CI59 

0.1343 

2.5400 

mined 

0.0099 

O.II83 

2.5729 

o;oii6 

0.1244 

2.555s 

I 


-C6-  - 


for  analysis^  v/as  discarded  without  its  total  ni  trogen  content  liaving  first  been 
determined. 

For  convenience^  the  results  on  the  distribution  of  nitrogen  in  the 
feed,  are  divided  into  nonprotein  nitrogen,  nitrogen  distribution  as  shown  by 
the  Van  Slyke  analysis,  and  the  nitrogen  lost  in  the  method  of  analysis. 

Criticism  has  been  made  of  the  application  of  the  Van  Slyke  method  to 
the  aiialysis  of  feeds  on  the  basis  of  the  possible  interference  of  some  of  the 
nonprotein  nitrogenous  constituents.  In  the  metnod  used  in  this  work  ariy  possi- 
ble interference  of  nonprotain  nitrogenous  compounds  is  completely  obviated  by 
the  complete  rauoval  of  those  compounds  by  extractions  with  ether,  shsolute 
alcohol,  and  cold  1 per  cant,  trichloracetic  acid.  Although  the  exact  role, 
played  in  protein  metabolism  by  all  of  the  nonprotein  rntrcgenous  constituents 
of  a feed,  is  at  present  unknown,  it  is  of  interest  to  note  that  the  four  feeds 
analyzed  vary  narkedly  in  their  ncnprotein  nitrogen  content.  Alfalfa  has  the 
highest  percentage  of  norprotein  nitrogen  with  an  average  of  19*09  cent., 
•while  cottonseed  meal  has  the  lowest  with  an  average  of  o.20  per  cent.  Oats 
and  corn  ijave  intermediate  values  of  1^.93  cent,  and  9*^3  psi*  cant.,  respec- 
tiveljr.  Judging  from  theso  values,  which  agree  vary  well  with  those  of  Grindley 
and  Eckstein  (Q),  roughages  have  a higiier  nonprotein  nitrogen  content  and  con- 
centrates a lower  content  than  the  cereals. 

jjnder  the  heading  ‘’nitrogen  lost  in  method  of  analysis'’  are  found  the 
results  of  the  total  nitrogen  determira.tions  on  the  various  fractions,  the 
amino-acid  content  of  which  escapes  analysis  by  the  method  of  preparing  the  hy- 
drolyzed protein  solution  and  by  the  Van  Slyke  analysis  of  this  hydrolyzed  pro- 
tein solution.  The  total  nitrogen  lost  is  shown  in  Column  U.  The  nitrogen 
lost  in  the  preparation  of  the  hydrolyzed  protein  solution  is  siiown  in  Column  0, 
representing  the  nitrogen  left  In  the  residue  after  treatment  ’with  the  last  ex- 
tracting fluid  - 5 per  cent,  sodium  hydroxide,  and  in  Column  P,  giving  the  nitro- 


1 


-63- 

gen  in  the  alcohol  precipitate  (starch)  of  the  hot  c per  cent,  trichloracetic 
acid  extract.  The  total  nitrogen  lost  in  the  preparation  of  the  hydrolyzed  pro- 
tein solution  v/as  0.2^9  cent,  in  ca.se  of  oats,  0.U12  per  cent,  in  case  of 
corn,  O.U3O  per  cent,  in  case  of  cottonseed  meal,  and  2.519  P®*  c®at.  in  case  of 
alfalfa.  The  extraction  of  starch  was  not  made  in  the  cases  of  cottonseed  meal 
and  alfalfa  because  of  the  sriall  amounts  present. 

The  nitrogen  lost  in  the  ar.alysis  of  the  hydrolyzed  protein  solution 
by  the  Van  Slyke  method,  consists  of  (0)  the  unadsorbed  hunin,  filtered  from  the 
amyl  alcohol-ether  aqueous  solution  during  the  decomposition  of  the  bases;  (E) 
the  nitrogen  extracted  by  the  amyl  alcohol-ether  mixture;  (S)  the  nitrogen  in 
the  residue  filtered  from  the  solution  of  the  bases;  and  (T)  the  nitrogen  in  the 
residue  filtered  from  the  solution  of  the  filtrate  from  the  bases.  As  percent- 
age of  the  total  nitrogen  of  the  feed  the  total  nitrogen  lost,  as  shown  in 
Column  U,  Table  I,  is  l.QO  per  cent,  in  case  of  oats,  3.ci9  cant,  of  cotton- 
seed meal,  3.65  per  cent,  of  corn,  and  Ij.73  P®!*  cent,  of  alfalfa. 

Tne  terms  used  in  the  literature  for  desigriating  the  humin  nitrogen 
fractions  are  confusing.  According  to  Van  Slyke' s original  method  (6):  "Dur- 
ing the  distillation  (of  ammonia)  e.ll  of  the  black  coloring  matter,  or  melanin, 
v;hich  is  formed  during  the  hydrolysis  of  the  proteins,  is  adsorbed  by  the  undis- 
solved lime."  The  latter  is  filtered  off,  washed,  and  submitted  to  Kjeldahl 
analysis.  The  results  are  reported  as  melanin  nitrogen.  This  is  the  only  frac- 
tion of  humin  reported  by  him.  In  the  first  s-ltempt  to  apply  the  Van  Slyke 
method  to  the  analysis  of  feeds,  Grindley,  Joseph,  and  Slater  (5),  used  the 
original  method  directly.  The  results  of  their  melanin  nitrogen  included, 
therefore,  any  nitrogenous  substances  in  the  insoluble  residue  of  the  f eed  a.s 
wall  as  the  melanin  or  humin  formed  duririg  the  hydrolysis.  ITollau  (7)  filtered 
off  the  insoluble  residue  remainirig  after  the  direct  hydrolysis  of  feeds  and 
analyzed  the  filtrate  according  to  tne  Van  Slyke  method.  His  results  v.’ere  based 


(»!. 


f ; ,. 


. X 


■ T*- 

F»: 


i ':<* 


■ *aV5Ji}-^  ''-,  -V'  j-i-« 

M 


m 


'.'mi 

''■fftj 

>•  ' 

^ Ki 


r.l 


''tfA  i--’;  'T.e^ 


.in 


H: 

t 


\ 

f .Ti-'. 


' .I'M 


iiv  . ... 


(:i 


' ; 


i 

r 


:S 


f*  .A? 


'ii/iM 


■f 

fi 


J . <a  y .j 


-,V;»’ 


./•  a'' 


< 


4 .'  -!•*■  -'■■••»  •*  .'.  fji.’  ■•  '.'.V^.v»  t 


i'^  /'lii. 


t'  ■•■li^ 

' V ‘Vx' 


'.  i<;  *r 


'i  T ’ 


I ■ .fi;,io.  ■. J tr-  'i>  i4  'ri  I'-vs 

. , ; *,.■:■  {»^r  . 


.■„■/•■  *;*.'■ 


■ /J 


V ■ /, 

V..' 


/■ 


. . . 1 


V i,  j ii.:v’j,s  ^<j  *A 

■ ..  !('-  -C*  i??- 


-6)4- 


on  the  percentage  of  the  total  viitrogen  in  this  filtrate  and  not  on  the  total 
nitrogen  of  the  feed.  Attention  v/as  called  to  this  fact  and  to  tho  introduction 
of  certain  errors  by  this  procedure  by  G-rindley  and  Slater  (6)  v/ho  refer  to  their 
melanin  fraction  as  humin^  which  is  probably  tne  better  r^ame  for  this  substarice. 
Gortner  (1^)  divided  the  humin  fraction  into  ••acid-insoluble"  and  "acid-soluble" 
(a-bsorbed  by  lime)  humin^  the  sum  of  the  two  being  the  total  hurnin  nitrogen. 
Gortner  and  Holm  (I3)  report  the  two  fractions  under  separate  headings  as  insol- 
uble humin  nitrogen  and  soluble  hurnixi  nitrogen.  Other  workers  have  reported 
this  humin  nitrogen  fraction  under  such  various  headings  as  ••h'un;in  N absorbed 
by  lime"  (Osborne^  Van  Slyke,  Leavenworth,  and  Vinograd,  "humin  nitrogen 

absorbed  by  magnesia"  (Miller,  3),  and  as  "melanin  nitrogen"  divided  into  "a  b c 
fraction"  and  "lime  fraction"  (Neidig  and  Sryder,  I7).  In  this  work  the  ®::- 
pressions  insoluble  and  soluble  humin  have  been  retained.  The  humin  nitrogen  of 
Table  III  is  the  sum  of  the  insoluble  and  the  soluble  humin  rhtrogen.  It  is 
possible  tixat  the  nitrogen,  or  parts  of  it  at  least,  in  the  fractions  listed  in 
Columns  Q,  B,  S,  and  T of  Tables  I and  II  properly  belongs  to  the  total  humin 
nitrogen  figures  but  this  has  not  been  done. 

Other  fractions  of  hurnin  nitrogen  have  been  reported.  In  Van  Slyke's 
improved  method  (^3)  of  decomposing  the  basic  phosphotungstates  bj'-  the  amyl 
alcohol-ether  method,  the  follov/ing  statement  is  made: 

"In  some  cases  the  aqueous  and  ether-amyl  alcohol  layers  do  not  sep- 
arate readily  v/ith  a clean  bomidary  between  them.  This  effect  is  due  to  the 
presence  of  e,  slight  amount  of  humin  which  may  loavs  escaped  previous  adsorption 
by  calcium  hydrate.  In  this  case  the  ur.adsorbod  humin  is  carried  dov/n  v/ith  the 
basic  phosphotungstates,  a,nd  fouls  the  solution  when  their  precipitate  is  de- 
composed as  above  described  (amyl  alcohol-ether  method).  In  order  to  clear  the 
solution  up,  it  is  all,  without  separation  of  the  aqueous  and  ether-amyl  alcohol 
layers,  passed  through  a Buchner  funnel  with  suction." 

It  is  evident  that  Van  Sl3/ke  recognized  a portion  of  humin  substances 
in  the  basic  phosphotungstates.  Osborne,  Van  Slyke,  Leavenworth,  and  Vinograd 
(25)  in  one  case  purified  their  basic  phosphotungstates  by  reprecipitation  with 


phoschc tungstic  acid.  Tney  decomposed  t..eir  basic  precipitate  by  the  amyl  alco- 
hol-other method  and  state  that  "all  the  coloring  matter  which  accompanied  the 
bases  was  extracted  by  the  amyl  alcohol  and  etner."  The  nitrogen  taken  up  by 
the  organic  solvents  was  determii:ied  and  reported  by  them  as  "humin  N in  amyl  alco- 
hol extract."  Gortner  ana  Holm  (I3)  separated,  besides  the  "acid-insoluble 
humin"  and  the  "acid-soluble  humin,"  a "phosphotungstic  humin."  This  ;vcis  de- 
termined by  submitting  the  barium  phos  oho  tungstate  precipitate'^  to  Kjelaahl 
analysis.  This  fraction  of  ijumin  nitrogen  is  undoubtedly  the  "humin  IT  in  amyl 
alcohol  extract"  of  Osborne,  Van  Slyke,  Leavenworth,  and  Vinograd  and  the  "un- 
adsorbed hurnin"  of  Van  Slyke.  ihis  fraction  is  again  divided  by  Miller  (3)  into 

"humin  IT  insoluble  in  amyl  alcohol"  and  "humin  iT  in  smyl  alcohol  extract."  Nei- 

dig  and  Sr^yder  (1])  also  found  tliat  a dark-colored  substance  usual_y  formed  along 
with  the  bases  when  the  latter  were  precipitated  with  phosphotungstic  acid  and 

refer  to  it  as  the  "phosphotungstic  humin"  of  Gortner  and  Holm.  Until  the  final 

method  wvs  adopted,  this  dark-colored  substance,  v/hich  v^as  sometimes  of  a sticmy 
nature,  was  often  er.countered.  With  this  substance  present  it  was  almost  im- 
possible to  wash  the  ba.sic  pliospho tungstates  thoroughly.  the  method  firally 
adopted  the  basic  phospho tungstates  of  all  four  of  the  feeds  analyzed  were  white, 
gray,  or  slightly  cresm-colored  gra-nula.r  precipitates  entirely  free  from  any 
dark-colored  material.  However,  during  the  decomposition  of  the  basic  phospho- 
tungstates  by  the  amyl  alcohol-etner  method,  a scum,  sometimes  of  a dark  color, 
is  formed  makings  filtration  necessary.  Tii  is  residue  is  washed  c5;-refully  with 
armnonia-free  water,  smyl  alcohol,  and  ether,  and  submitted  to  Kjeldahl  analysis. 
The  resLilts  are  reported  in  Column  Q under  the  heading  "UTiadsorbed  humin,"  the 
nsme  used  by  Van  Slyke.  The  clear  aroyl  alcohol-ether  extract  of 

^ These  authors  used  Van  Sijhce's  original  barium:  hydroxide  method  of  liber- 
atine  the  basses. 


,1' 

'J 


I 


I - ^ . - 


'*'■■  r -.Y^' : - < ■[,'. 

r 

y 


I 


j"  ’■■  'i, . ■> 


T .:•  f 


1^, , i »■ 


<KS‘A 


/f 

-OD- 

tr-e  phosphot’angstic  acid  is  also  subaiitted  to  Kjeldahl  aralysis  and  tne  nitro- 
gen content  reported  n*  Colmm  R as  the  nitrogen  in  axnyl  alcohol -ether  extract. 

On  concentrating  the  solution  of  the  bases  a small  gray  residue  has  been  found 
to  settle  out.  Van  Slyke,  in  the  original  method  of  freeing  the  bases  by  means 
of  barium  hydrate,  stated  that  this  residue  v/as  barium  pliospho tungstate,  which 
was  to  be  filtered  off  and  evidently  discarded,  Tliis  residue  has  alwaj/s  been 
encountered  hi  this  v/ork  even  when  the  amyl  alcohol-ether  method  of  decomposing 
the  basic  phosphotungstates  v;as  employed.  According  to  the  detailed  procedure 
this  residue  is  filtered  off,  washed  carefully  and  its  total  nitrogen  determined 
Tile  results  are  shown  in  Column  S.  After  making  the  filtrate  from  the  bases  up 
to  volume  (tlOO  cc.)  ind  allov/ing  to  stand  over  night  occasionally  a residue, 
similar  to  the  one  in  the  solution  of  the  ba.ses,  separe^tes  out.  This  is  usually 
very  small  in  amount,  but  when  present  the  nitrogen  has  been  determined.  The 
results  are  shov/n  in  Column  T,  vo  attempt  has  been  made  to  determine  the  nature 
of  the  nitrogenous  constituents  in  these  fractions.  The  total  nitrogen  has  been 
determined  in  than  entirej-y  for  the  purpose  of  showing  the  accura-cy  or  inaccuracj 
of  the  method  as  applied  to  the  aioalysis  of  feeds. 

As  percentage  of  the  total  nitrogen  of  the  feed,  the  humin  ri  trogen  oi 
oats  is  cent.,  of  corn  3.5^  psr  cent.,  of  cottonseed  ineal  0.3O  per  cent, 

and  of  alfalfa  ].}6  per  cent. 

The  completeness  with  which  the  nitrogen  is  extracted  from  the  finely 
ground  feeds  is  shown  in  Column  P,  Table  I,  which  gives  the  percentages  of  the 
total  nitrogen  of  the  feeds  remaining  in  the  residues  a-fter  the  last  extra.ction 
with  5 per  cent,  sodium  hydr&xide  solution.  The  nitrogen  in  the  residue  from 
oats,  as  shovm  by  the  a-verage,  is  0.13^  per  cent,  of  the  origir.al  amount  present, 
from  corn  0,136  per  cent.,  from  cottonseed  meal  O.U3O  per  cent.,  and  from  al- 
falfa £.519  per  cent. 


The  nitrogen  distribution  as  shovv-n  by  the  Van  Slyke  ar^ilysis  includes 
the  insoluble  huiain  nitrogen,  the  soluble  huniin  nitrogen,  acid-air.ide  nitrogen, 
arginine  nitrogen,  cystine  nitrogen,  histidine  nitrogen,  lysine  nitrogen,  amino- 
acid  nitrogen  in  the  filtrate  from  tne  bases,  and  the  non-amino -acid  nitrogen 
in  the  filtrate  from  the  bases.  Tiie  question  has  been  raised  in  several  papers 
on  the  analysis  of  the  proteins  of  feeds  by  the  Van  Slyke  method  that  the  various 
fractions  ordiriarily  designated  "arginine  nitrogen,"  "histidine  nitrogen,"  etc., 
may  not  be  accux’ately  described  by  these  terms,  on  account  of  the  heterogeneous 
nature  of  the  nitrogenous  constituents  as  well  as  other  substances  present.  In 
the  total  absence  of  exqxerimental  evidence  on  this  point,  it  seams  fair  to  assume 
the.t  the  above  terms  are  as  properly  applied  to  the  fractions  of  nitrogen  ob- 
tained in  the  analysis  of  feeds  by  the  above  described  method  as  to  the  corres- 
ponding fractions  obtained  in  the  analysis  of  pure  proteins. 

It  is  of  interest  to  note  the  variation  in  the  basic  lutrogen  of  the 
different  feeds,  since  v/e  knov/  more  of  the  requirements  of  animals  for  the  basic 
amino-acids  tlxan  for  a.ny  other  group,  line  argirdne  nitrogen  varies  from  about 
d per  cent,  of  the  total  nitrogen  in  alfalfa  to  18.]  per  cent,  in  cottonseed 
meal.  The  cystine  nitrogen  is  about  the  same  in  ail  cases  but  these  results 
ma^/,  perhaps,  be  a little  lov/.  The  histidine  nitrogen  is  lowest  in  alfalfa  with 
8.  value  of  3.9  Por  cent,  of  the  total  nitrogen  and  cottonseed  meal  is  highest 
with  a value  of  ].£  per  cent.  Lysine  rdtrogen  is  lowest  in  corn  with  a "value  of 
ci.c:  par  cent.,  sli^tly  higher  in  oats  with  d.8  per  cent.,  and  highest  in  alfalfa 
with  U.43  pel*  cent.  The  total  basic  nitrogen  as  percentage  of  total  nitrogen 
is  31. 03  per  cent,  in  cottonseed  meal,  cl.83  per  cent,  in  oats,  1]*35  cent, 
in  alfalfa,  and  lb. 83  par  cent,  in  corn. 

In  Table  III  the  average  results  obtained  in  Table  I are  compared 
with  the  results  on  the  same  feeds  obtained  by  Grindlay  and  associates  reported 
by  Grindley  (14)  and  by  ITollau  (7). 


„ ^ J U 


a 


1 


V 


i;,  . *.i  .f  ,<>v  ,y  ;. 


■I'jf  7,h  Si- 
V 


- ’,wj'  V V 


• ' • *v» 


•‘“W  ¥ ■ 

'■  . \ « 

. j * ' ’ 

1^*  V ,; 


£'■’ •'■ 


*\> 


'V/ti 

4- 


4 ■ ' v‘  • 


* "'  - ’•■;  -s  ".  .>:•  r,;  .'2;>  ,-i^  ;^j 

■■'  , , • - ,■ 


< ' 


»fk..  r.’ 


•‘'  *-.  V • ' ,'  ' 

. '*«. 


r-^ 

!l  i •; .’  fti'  \ ■ 


' ■•■'  i 


■"J  ■■ 


♦ .'■■  ♦' 


-f  ■*.»■.> 


■ i 


'W'r-'- 


y} 


y 


'U  ».'■  •■ 


¥■ 


■ ■?•/-:.  vv.  ,. 

-I  ■ .■  ' , *' 


'v  ‘ ' J 

■ 'Wl'*'"'':  jra 


■>  •:< 


• kJ 


,.  S*-'4 

' ■'  ' ’.  I '■  J*T»  T'X' 


M I 


' , . y*  ' ■*  ■'  \ ' , I * »‘  ^ . ' ...  • '.i 


• V*-»v 


vi : h ’i,’^^  ' 


■.i  .';''',n.  V ■'.'i 


m. 


TABLE  III.  - G0:,1PAEIS0N  OF  RESULTS  WITH  THOSE  OF  PREVIOUS  IHVESTIGATORS 
(Results  expressed  as  percentage  of  total  nitrogen) 


■^IbT 

-OOSS’B  pu-0 

iCsip  'T.lp 


I 

eiq-Gj, 

uiojj; 


TIB  I TOE 


5lbT  se^BT 

-OOSSB  pUB 


I 

raoj^ 


TIBI  TOE 


^Ibl  BBYSX 
-OOSSB  pUB 
iCsipuTai) 


I 

tnojji 


n.BiTOE 


5lcl  seqBt 

-OOSSB  piTB 

iCoXpuiap 


I 

STOBJ, 

U!0 


n. 


Ph 

< 


Pi 

W 

CO 

o 

o 

o 


s 

o 

o 


CO 

EH 


-oo- 

=}•  (S'<  O O -=J  O VI  O" 
i — > O o -=T  .-H  O I — 


O U'l  i'-O  I— -U-  J O 


"!y 

r-i 

Y) 

O 


'£>  O ex'  r-H  U ^ J- 

fH^  O (T-  O'  -=t  o LPi  U > X)  '-O  I'-  VJ 


'VD 


O O O lU  O r-l ' o ^ 

rH 


O 


'-o  I-—  I'-  -=!■  f — ^ .r\j  O'' 

O U 1— o -=1' 


'■O  ,vj 


u^. 

-=T 


vD 

■O 


O' 


Li  ^ 'O  -U  LOv  1^ — 'O  C\J  I 
^ i'~  lI'>vO  -4"  X)  ^ I 

• • ••••••!• 

O i — 0^  O -=t  \J  LT\  I 
f-H  <-4  I 


O 

O' 

v6 

cr. 


rH  O 

rH 

i-H 

3 O 

O'.  35  cr 

NX 

3-  NX 

i'-  O' 

rH  3 

^ rH 

ax  ax  3 

-rO 

C5X  vO 

CO  o 

r*H 

I-— 3' 

Q 3 O 
3 

o ax  NX 

cr 

ox 

NX  o crx.o  ox  NX  cr  o 

XO  o rH  O 3 axvX)  o 

1 

1 

1 

1 

1 

1 

1 

1 

ox 

ox 

3 . — V O 3 3 O cr  . O 

r— f 

1 

1 

1 

1 

1 

1 

1 

1 

cr- 

fH'y.' 

U'> 


■ 0>  X)  O l^'^O  I' 

LTtrH  -U  r- 


u CTN  X)  cvj  roi-t  oj  I 

r-l  LTi, 


COi 


r— I 

o 


^ ^ K>0  O C\J  IT'*  r\J 

O'.  Lf O 50  CVJ  S'—  '-U  KX  i-H  CO  OX 


rH  CO  rH  -:d-  rvj  '^O  I' 
rH 


■o  rH  "X) 


ox 

O' 


iH  ,\J  CQ  'O  O ox  O'  1 

Kx  o \ Br  -3-  ax  o -H'  c\j  i 

I 

OJ  rH  -3  o O fHX  rH  I 

tH  .H  J-  rH  I 


3" 


vD 

O' 


'-O  -3  xj  '-D  f\l  cx  rvj  o I 

O o.  3hh  ^X-3  r—  ox  I 

I 

NX  O'.  rH  rH  -3  KX  rH  rr-  » 
r-H  rH  UX  I 


KX 

O 


NX  OX-3  o -3  3-  VX>  I'-  -3  NXO 
aXvD  ox  ro  !X)  IH  CO  OX  3 rH  O' 


rH  OX  rH  O irx  Al 


NXO  rH  rH  rH 

I — i 


o 

o 

cr. 

O' 


W 

05 

0) 

R; 

ca 

rO 

o 

0) 

<D 

P 

•H 

Oi 

a 

•.H 

CA 

qJ 

o 

>L 

rO 

p 

rH 

a 

Vi 

Tv 

o 

• 

•H 

p 

O 

'H 

+3 

I — 1 

rH 

r-l 

o 

• 

•rl 

O 

P 

o 

•rJ 

+> 

(0 

o 

r-H 

a 

rH 

3 

XI 

•H 

o 

H3 

■+H 

rH 

u 

(0 

3 

rH 

e 

O 

I— » 

<D 

*iH 

O 

rH 

lO 

• 

rH 

<D 

sd 

1**:^ 

o 

•iH 

cd 

(Sj 

<D 

•fH 

r-H 

•»H 

cn 

rH 

4J 

•H 

•rl 

(D 

s 

O 

rH 

-M 

o 

•iH 

•H 

a 

o 

p 

.4 

•H 

C/3 

Eh 

q 

*rl  *ri 

43 

•rH 

sd 

1 

05 

o 

Cm 

O 

a 

a bo 

CA 

(/) 

M 

nH 

3 

o 

c — 1 

•iH 

s 

o 

4.3 

1 — 1 

Pd  H 

o 

3 

HH 

rr 


-69- 

As  a whole,  the  determinations  of  the  different  investigators  do  not 
agree  well,  althoudi  in  some  instances  the  agreement  is  quite  satisfactory. 

The  results  reported  in  Table  I agree  with  those  of  Grindley  and  associates 
slightly  batter  than  they  do  with  those  of  IJollau.  The  lack  of  concordant  re- 
sults is  due,  chiefly,  to  differences  in  methods  used.  Grindley  and  associates 
hydrolyzed  the  finely  gro-ond  feeds,  determined  the 'acid-airi de  nitrogen  in  the 
hydrolysate,  filtered  off  the  hurain  nitrogen,  and  proceeded  to  analyze  the  fil- 
trate according  to  the  Van  Slyke  method.  Their  results  were  based  on  the  total 
nitrogen  in  the  feed.  Noliau  removed  the  fat  by  extracting  the  finely  ground 
feed  witn  ether.  The  sairples  v/ere  then  completely  hydrolyzed  and  the  insoluble 
humin  filtered  off.  The  total  nitrogen  determined  in  the  filtrate  was  the  basis 
for  calculation  of  results  of  the  -nitrogen  distribution  a-s  obtained  "by  the  Van 
Slyke  procedure.  By  the  iinproved  method  described  above  the  nonprotein  nitrogen- 
ous constituents,  most  of  the  carbohydrates  and  the  fiber  are  rs-noved  before 
hydrolysis.  The  objectionable  features  of  the  previous  methods  are  thereby  ob- 
viated completely  or  at  least  greatly  reduced.  Considering  the  differences  in 
procedure,  Nollau's  results  for  humin  nitrogen  should  be  lov/er  than  the  results 
for  the  humin  nitrogen  of  Grindley  and  a.ssociates.  In  genvsral  this  is  true. 
Nollau's  procedure  should^also  lead  to  correspondingly  higher  results  for  the 
remaining  nitrogen  values,  considered  on  the  basis  of  the  total  nitrogen  of  the 
feed.  V/itli  a few  sxceptiais  this  is  found  to  be  true,  but  the  results  are  usu- 
ally higher  than  this  difference  of  procedure  would  warrant.  The  lower  results 
for  armconia  nitrogen,  the  amino-acid  nitrogen  and  non-amino-acid  nitrogen  in  the 
filtrate  from  the  bases  of  the  method  used  in  this  work  are  expected  because  of 
the  removal  of  the  nonprotein  nitrogen.  There  a,re  some  differences  in  the  re- 
sults tiiat  cannot  be  e^splained  on  the  basis  of  differences  of  procedure.  For 
example,  Nollau  reports  no  lysine  in  oats  v\foile  2.84  psr  cent,  was  found,  he  re- 


/ 


1 


-70- 


ports  6.53  per  cent,  of  lysine  in  corn  while  only  c..'d  per  cent,  w&s  fo'und,  and 
again  he  reports  no  non-aniino-acid  nitrogen  in  the  filtrate  from  the  bases  in 
com  while  ']  ,'d.cL  per  cent,  was  found.  The  results  IToliau  obtained  for  cystine 
are  in  all  cases  much  higher  than  those  reported  in  Table  I but  it  is  very  prob- 
able that  the  cystine  nitrogen  reported  in  Table  I is  lass  than  half  the  origir.al 
amount  present. 

Miller  (3)  in  his  study  of  the  distribution  of  nitrogen  in  the  alfalfa 
seed  precipitates  the  protein  from  a O.5  P52*  cent,  potassinm  hydroxide  extract 
with  acetic  acid  and  arialyses  the  precipitate  which  contains  only  bO  per  cent, 
of  the  total  nitrogen  of  the  seed  according  to  the  Van  Slyke  ara.lysis.  The  re- 
sults obtained  therefore,  cannot  be  considered  as  representing  the  distribution 
of  nitrogen  in  the  entire  seed.  Dowell  and  Menaul  (I4)  report  on  the  "Nitrogen 
Distribution  of  the  Proteins  Extracted  by  Dilute  Alkali  from  Pecans,  Peanuts, 
Kafir,  and  Alfalfa."  Their  method,  \'\4iicji  was  similar  to  that  used  by  Miller,  v/as 
in  case  of  alfalfa  as  follows:  "Alfalfa  which  was  ground  to  pass  a l|0-mesh  sieve 
was  extracted  with  O.3  per  cent,  sodium  hydroxide,  and  62  per  cent,  of  the  nitro- 
gen compounds  were  extracted.  Sixty  one  per  cent,  of  the  rhtrogen  extracted  was 
precipitated  v/hen  the  solution  was  inade  sli^tly  acid  with  acetic  acid.  The  puri- 
ty of  the  precipitated  proteins  was  found  to  be  85  per  cent.,  using  the  factor 
b.25«" 

The  results  of  Table  I on  alfalfa  are  compared  with  those  of  Dowell 
and  Menaul,  in  Table  IV.  Tiio  agreement  is  obviously  very  poor. 


-71- 

TABLE  IV.  - NITBOGEI'I  DISTRIBUTION  OF  ALFALFA 


Dov/ell  and 
Menaul'^ 

From 

Table 

IvlioN 

S.SO 

S.17 

Him:  in  N 

7. SO 

7-3S 

Arginine  N 

11.01 

6.00 

Histidine  N 

6 .h& 

3 -^'3 

Cystine  N 

0.65 

o.qQ 

Lysine  N 

'do 

4.43 

Mono-anino  N 

53-^3 

33.03 

Non--amino  N 

s . 4s 

> C*! 

^ Average  of  ci  analyses;  results  expressed  as  per- 
centage of  total  nitrogen  of  the  protein  prep- 
aration. 

^0  Average  of  4 analyses;  results  expressed  as  per- 
centage of  total  nitrogen  of  the  alfalfa. 

‘ Dowell  and  llenaul  rfiake  the  ststement:  "We  Imve  taken  advantage  of 

the  fact  that  all  proteins  are  soluble  in  basic  solutions  to  separs.te  them  from 
the  other  subste.nces  in  foods  and  feeds  which  make  it  impracticable  to  apply 
the  Van  Slyke  method  to  determine  the  nitrogen  distribution."  While  the  precip- 
itated proteins  in  case  of  pecans  and  peanuts  represented  a slightly  larger  per- 
centage of  the  total  nitrogen  of  the  food  stuff,  the  proteins  precipitated  from 
the  alkali  extract  of  alfalfa  represented  less  than  40  psr  cent,  of  the  total 
nitrogen  of  the  alfalfa.  Evidently  a considerable  fraction  of  the  proteins  of 
these  vegetable  naterials  are  net  soluble  in  basic  solvents.  Yet  these  authors 
conclude  that  "the  extractions  of  the  proteins  v.uth  dilute  alkaline  solutions 
may  enable  us  to  obtain  the  araino-acid  composition  of  foods  and  feeds  by  means 
of  the  Van  Slyke  method."  With  such  large  percentages  of  the  nitrogen  of  a 
food  or  feed  remaining  in  the  unauialyzed  residue,  the  significance  of  the  re- 
sults obtained  on  the  extracted  proteins  may  be  seriously  questioned  and  it  is 
doubtful  whetner  the  proteins  of  feeds  may  be  quantitatively  separated  from  the 
other  constituents  by  any  such  simple  procedure  as  this. 


-7^- 

VIII.  SU!.:,1AEY  AND  CONCLUSIONS 

1.  A ruethod  has  been  developed  for  treating  a sample  of  feed  so  that 
the  proteins  which  it  contained  are  obtained  in  solutions  sufficiently  free  from 
interfering  substances  so  that  the  Van  Slyke  method  for  the  determination  of  cer- 
tain amirh-acids  in  purs  proteins  may  be  applied. 

c.  The  completeness  vath  v;hich  this  treatment  removes  the  nitrogen 
from  the  finely  ground  feed  v/as,  as  shovm  by  the  avera.ges,  per  cent,  of 

the  oats,  99*^o4  poi*  cent,  of  the  corn,  99*570  per  cent,  of  the  cottonseed  meal, 
and  97*4o1  per  cent,  of  the  alfa.lfa. 

3.  The  objectionable  parts  of  previous  procedures  for  the  applica- 
tion of  the  Van  Slyke  method  to  the  determirjation  of  amino-acids  of  feeds  have 
been  obviated  coinplstely  or  at  laast  greatly  reduced  by  the  following  features 
of  the  method  used  in  this  work: 

(aj  The  nonprotsin  nitrogenous  constituents  are  removed  by  extractions 
v/ith  absolute  ether,  cold  absolute  alcohol  and  cold  1.0  per  cent,  tricliloracetic 

^ I 

acid. 

(b)  The  starch  is  removed  by  a hot  2.0  per  cent,  trichloracetic  acid 

extra  ction. 

(c)  The  fiber  is  not  present  during  the  hydrolysis  of  the  proteins. 

4.  The  amino-acid  contents  of  oats,  corn,  cottonseed  mee.l,  and  al- 
falfa, as  determined  by  the  Van  Slyke  Method,  a-re  reported. 

5.  By  further  application  of  available  methods  for  the  estimation  of 
other  amino-acids  to  hydrolyzed  protein  solutions,  prepared  in  a manner  similar 
to  that  described  for  this  work,  it  may  be  possible  to  obtain  further  important 
knowledge  concerning  the  nutritive  value  of  the  proteins  of  foods  and  feeds. 


. ....  . 

j^j  ,v.’-' 

4*-’* 


■ ■ ’ ■ r-:^.  'w:m 

i !^<^.,<i;?'-\  u'«5,.4«. /.  :,-)i,?«  ■.  ii  ,4«Kii  » ti 

f .. j--;  ■ I 

^.r.  ; '••  ^:  rf:'4m?:/.^*;'.i,- ^ 

h' ■■  ■■' ' ' • •‘-.A’.  • •■ '..'  ••  ^'  Mi 


k'  >l^':ijSfi|(Mr>^^  •■»«-•■  » ^ ■ «r>^SiaBP^.R-'-  ^ 

.•■  , .:.■  '.-. . ■ • >.  % :^^L'  ..:v  V*:™  » 


JL  y ..  » ••.  V ' -V  . > -.  ■ </n,.  '..•  • , ''.,<'5.  >1 »«/ ‘ .. 

I •■  , :„  t- . ;? , C,,>— .,:  , 

Jfv  -Vi,-.*  ,->i.-.  >.. .’  ■ -Mi*  W-  k]^.  >'-^m\. 


h iKi-  •V'  " '^." /^  ■ ■■'  \^M  M 

j-,,.  >;i- 

SiilEsnt: 

in^ 


V...*  ..'iafi’if 

■■  -r  .rViiMi 


''■4(&' 


: ; 4;, 


ilJ'? 


(i.'.'.JtJi'*^  " '•v^  ' ..Alii..*’ V-.' 


-73- 


IX.  BIBLIOGRAPHY 

(1)  Osborne,  T.  B.,  The  Vegetable  Proteins,  1909,  13* 

(H)  Osborne,  T.  B.  and  Mendel,  L.  B.,  J.  Biol.  Chear.,  1914,  xviii,  1. 

(3)  Miller,  H.  G.,  J.  Aiu.  Chem.  Soc..  1921,  xliii,  OOo. 

(4)  Dowell,  C.  T.  and  Menaul,  P.,  J.  Biol.  Chem..  1921,  xlvi,  437* 

(5)  Grindley,  H.  S.,  Joseph,  W.  E.,  and  Slater,  M.  E.,  J.  Ann  Cheiri.  Soc..  1915, 

xxxvii,  177^* 

(6)  Van  Slyke,  D.  D.,  J.  Biol.  Chem.,  1911-12,  x,  I5. 

(7)  Hollau,  E.  H.,  J.  Biol.  Chem..  1915>  xxi,  6II. 

(S)  Grindley,  H.  S.  and  Slater,  M.  E.,  J.  Am.  Chem.  Soc.,  1915>  xxxvii,  c'(ocl. 

(9)  Grindley,  H.  S.  and  Eckstein,  H.  C.,  J.  Am.  Chem.  Soc.,  1916,  xxxviii,  1425. 

(10)  Hart,  E.  B.  and  Bentley,  W.  H.,  J.  Biol.  Chern.,  1915^  xxii,  477* 

(11)  Gortner,  R.  A.  and  Blish,  M.  J.,  J.  An.  Chem.  Soc..  1915^  xxxvii,  I63O. 

(12)  Gortner,  R.  A.,  J.  Biol.  Chem..  1916,  xxvi,  177* 

(13)  Gortner,  R.  A.  and  Holm,  G.  E.,  J.  Am.  Chem.  Soc.,  I917,  xxxix,  2477* 

(14)  Grindley,  H.  S.,  Proc.  Am.  Soc.  Animal  Production.  I916,  l33* 

(15)  Eckstein,  H.  C.  and  Grindley,  H.  S.,  J.  Biol.  Chem.,  1919>  xxxvii,  373* 

(16)  Hartley,  P.,  Biochem.  J.,  1914,  viii,  541. 

(17)  Neidig,  R.  E.  and  Snyder,  R,  S..J.  Am.  Chem.  Soc.,  1921,  xliii,  951* 

(lii)  Greenwald,  I.,  J.  Biol.  Chem..  1915,  3cxi,  6I;  Ibid.,  191&^  xxxiv,  97* 

(19)  Folin,  0.  and  Denis,  W.,  J.  Biol.  Chem..  I916,  xxvi,  491* 

(20)  Wolff,  C.  G.  L.,  J.  Physiol..  1Q15>  xlix,  S9. 

(21)  Hill,  R,  L.,  J.  Biol.  Cha^i..  1915,  xx,  175* 

(22)  Van  Slyke,  D.  D.,  Vinograd-Villchur , M.,  and  Losee,  J.  R.,  J.  Biol.  Gnem.. 

1915.  xxiii,  377. 

(23)  Van  Slyke,  D.  D.,  J.  Biol.  Chem..  1915,  xxii,  2S1 . 

(24)  Winterstein,  E.,  Ze i t s ciir . f . pby s i 0 1 . Che;n . . 1901-02,  xxxiv,  I53. 

(25)  Osborne,  T.  B.,  Van  Slyke,  D.  D.,  Leavenworth,  C.  S.,  and  Vinograd,  M., 

J.  Biol.  Chem..  1915^  xxii,  259. 


• ■ V V.- 


"P-  . 


•y  ..N 

/ 

N 


- >,■  • • ) ■ 


:;  ' 'V  . > 


^ fr 


% 

■ i" 


• -/r.  ^■'  ■ 

• II 

-cW  ’.'  '■  i ■ • 


V 


»■  ' 


■ '-ii 


. 'AV- 
>/ ; ■ 


* - ^5.  ^ ■ V 


fr-'*-'.  . ’ V •■  . 

^ - :.  rr. 

y . . ,■’, 

* *"  -V‘v 

*V1  , : 


r. 


